From c0e30b69d11c41831c8d3426cea1f85de629890b Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Sun, 2 May 2021 00:19:46 +0000 Subject: [PATCH 01/21] Small fixes --- scripts/ds_extract_hc_pos.py | 2 +- scripts/organize_by_chr.py | 2 +- scripts/shuffle_data.py | 4 ++-- scripts/subsample_fast5.py | 4 ++-- src/extract_features.py | 8 ++++---- src/inference.py | 3 +++ src/utils/bed_processing.py | 2 +- src/utils/datasets.py | 4 ++-- src/utils/models.py | 18 +++++++++--------- src/utils/writer.py | 3 +-- 10 files changed, 26 insertions(+), 24 deletions(-) diff --git a/scripts/ds_extract_hc_pos.py b/scripts/ds_extract_hc_pos.py index adc6bbd..a4dab46 100644 --- a/scripts/ds_extract_hc_pos.py +++ b/scripts/ds_extract_hc_pos.py @@ -39,4 +39,4 @@ def create_arguments(): if __name__ == '__main__': args = create_arguments() - extract_hc_pos(args.bed, args.nomod, args.mod) \ No newline at end of file + extract_hc_pos(args.bed, args.nomod, args.mod) diff --git a/scripts/organize_by_chr.py b/scripts/organize_by_chr.py index a7ce744..de3b984 100644 --- a/scripts/organize_by_chr.py +++ b/scripts/organize_by_chr.py @@ -84,13 +84,13 @@ def write_mappings(mappings: Dict[str, List[Path]], out_folder: Path) -> None: mapping_f.write(f' {str(p)}\n') - def get_read_id(path): with h5py.File(path, 'r') as f: read_name = list(f[FAST5_READS_PATH].keys()) read_group = f[f'{FAST5_READS_PATH}/{read_name}'] return read_group.attrs['read_id'] + def organize_fast5(fast5_path: Path, align_path: Path, out_folder: Path, recursive: bool = False) -> None: align_data = {} counter = Counter() diff --git a/scripts/shuffle_data.py b/scripts/shuffle_data.py index 7cc00fe..bc809e4 100644 --- a/scripts/shuffle_data.py +++ b/scripts/shuffle_data.py @@ -105,7 +105,7 @@ def create_args(): check_size_parser.add_argument('-i', '--input_path', type=str, required=True) - #Shuffle command + # Shuffle command shuffle_parser = subparsers.add_parser('shuffle', help='Shuffle dataset.') check_size_parser.set_defaults(command=shuffle) @@ -129,4 +129,4 @@ def create_args(): if __name__ == '__main__': args = create_args() - args.command(args) \ No newline at end of file + args.command(args) diff --git a/scripts/subsample_fast5.py b/scripts/subsample_fast5.py index d3f6018..e08b6f3 100644 --- a/scripts/subsample_fast5.py +++ b/scripts/subsample_fast5.py @@ -4,7 +4,7 @@ import shutil from tqdm import tqdm -#import tqdm.contrib +# import tqdm.contrib from typing import * @@ -60,7 +60,7 @@ def sample_fast5(args): tqdm.write(f'{len(files)} files for sampling.') - # create output dir + # Create output dir try: args.out_folder.mkdir(parents=True, exist_ok=False) except FileExistsError: diff --git a/src/extract_features.py b/src/extract_features.py index 34b8863..13962cd 100644 --- a/src/extract_features.py +++ b/src/extract_features.py @@ -91,11 +91,11 @@ def process_read( sample_size: int=20, window: int=8, bed_pos: Optional[Set[GenomicPos]]=None) -> Optional[FeaturesData]: - """ This function process the given read to generate data. + """ This function processes the given read to generate data. - This function process extracts resegmentation information, samples the signal and extracts alignment data. + This function extracts resegmentation information, samples the signal and extracts alignment data. - :param path: Path to the signle FAST5 read + :param path: Path to the single FAST5 read :param reseg_path: Resegmentation path in re-segmented FAST5 file :param norm_method: Signal normalization method :param motif: Motif for which positions will be extracted @@ -145,7 +145,7 @@ def init_workers( def worker_process_reads(paths: List[Path], out_path: Path) -> Tuple[Path, int]: - """ Function that process input file list and stores generated data + """ Function that processes input file list and stores generated data :param paths: List of input files that will be processed :param out_path: Path to the generated output diff --git a/src/inference.py b/src/inference.py index ff059df..5a76a73 100644 --- a/src/inference.py +++ b/src/inference.py @@ -163,6 +163,7 @@ def test(args): out.write(tmp_f.read()) os.remove(worker_path) + def get_arguments(): parser = argparse.ArgumentParser() parser.add_argument('checkpoint', type=str) @@ -192,10 +193,12 @@ def get_arguments(): return parser.parse_args() + def main(): args = get_arguments() test(args) + if __name__ == "__main__": main() diff --git a/src/utils/bed_processing.py b/src/utils/bed_processing.py index 69b3d4d..2206d9a 100644 --- a/src/utils/bed_processing.py +++ b/src/utils/bed_processing.py @@ -47,7 +47,7 @@ def high_confidence_filter( that has coverage of at least 10 reads and having all reads modified or unmodified (modification frequency of 0% or 100%). - :param genomic_pos: Genomic positions that is tested. Not used + :param genomic_pos: Genomic position that is tested. Not used :param mod_info: Modification information for the given positions :return: True if position is high confidence position, False otherwise """ diff --git a/src/utils/datasets.py b/src/utils/datasets.py index ab8dc15..b89711b 100644 --- a/src/utils/datasets.py +++ b/src/utils/datasets.py @@ -17,7 +17,7 @@ def dataset_size(path): length, r = divmod(os.path.getsize(path), DATA_DTYPE.itemsize) - assert r == 0, 'Cannot calculate number of examples. Possibly wrong data file.' + assert r == 0, 'Cannot calculate a number of examples. Possibly wrong data file.' return length @@ -109,7 +109,7 @@ def val_dataloader(self): pin_memory=True, worker_init_fn=worker_init_fn) @classmethod - def add_argparse_args(cls, parent_parser) : + def add_argparse_args(cls, parent_parser): parser = argparse.ArgumentParser(parents=[parent_parser], add_help=False) parser.add_argument('train_path', type=str) diff --git a/src/utils/models.py b/src/utils/models.py index 26e079c..721dd54 100644 --- a/src/utils/models.py +++ b/src/utils/models.py @@ -43,7 +43,7 @@ def strand_from_str(string: str) -> Strand: @dataclass(eq=True, frozen=True) class GenomicPos: - """ Dataclass that describes genomic position. + """ Data class that describes genomic position. Genomic position is uniquely defined with chromosome, position on the chromosome and strand. """ @@ -54,9 +54,9 @@ class GenomicPos: @dataclass(frozen=True) class ModInfo: - """ Dataclass that describes modification information. + """ Data class that describes modification information. - Dataclass contains information about coverage and modification frequency (usually for genomic position). + Data class contains information about coverage and modification frequency (usually for genomic position). """ n_reads: int mod_freq: int @@ -153,7 +153,7 @@ def __init__(self, fd: h5py.File, normalization: Optional[str]=None) -> None: def get_read_id(self) -> str: """ Returns read id - :return: read id for given single FAST5 file + :return: read id for the given single FAST5 file """ return self.fd[self.read_group_path].attrs['read_id'].decode() @@ -174,7 +174,7 @@ def get_raw_signal(self, continuous: bool=True) -> np.ndarray: Returns the raw signal for the given read. Optionally, it converts discrete signal to continuous signal. - :param continuous: True if returned signal should be continuous, otherwise false + :param continuous: True if returned signal should be continuous, otherwise False :return: Raw signal for the given read """ @@ -190,7 +190,7 @@ def get_raw_signal(self, continuous: bool=True) -> np.ndarray: def signal_for_interval(self, interval: Interval, samples: int=20) -> Tuple[np.ndarray, np.ndarray]: """ Function that samples signal points from the given interval. - This function is used for sampling singal points from the given interval. If desired number of points is lower + This function is used for sampling signal points from the given interval. If desired number of points is lower than number of points in the given interval, some points are discarded. If desired number of points is higher, some of the points are repeated. Sampling is done by finding point indices using numpy linspace function. @@ -264,7 +264,7 @@ def map_signal_to_bases(self, points_indices: Set[int]=None) -> Dict[int, Baseca points_to_data = {} - start, base_index = self.relative_start, 2 # we take quality for central base + start, base_index = self.relative_start, 2 # We take quality for central base for event_data in self.event_table: event_length = int(event_data['length'] * self.read.sampling_rate) end = start + event_length @@ -353,9 +353,9 @@ def get_relative_to_raw(self) -> int: return self.read.fd[self.events_path].attrs['read_start_rel_to_raw'] def motif_positions(self, motif: str='CG', window: int=8, bed_pos: Set[GenomicPos]=None) -> Set[Interval]: - """ Function returns intervals that correspond to given motif. + """ Function returns intervals that correspond to the given motif. - This function extracts intervals that contain given motif. Start of the motif is the central + This function extracts intervals that contain the given motif. Start of the motif is the central index in the interval. If genomic positions are provided, only positions included in the given set will be returned. diff --git a/src/utils/writer.py b/src/utils/writer.py index e150f5f..1f2f4d0 100644 --- a/src/utils/writer.py +++ b/src/utils/writer.py @@ -47,7 +47,6 @@ def __init__(self, filename: str, generation_info: Dict[str, Any]) -> None: self.filename = filename self.generation_info = generation_info - def __enter__(self): self.fd = h5py.File(self.filename, 'w', libver='latest') @@ -153,7 +152,7 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label @staticmethod def on_extraction_finish(*args, **kwargs) -> None: - """Function that concatenates temporary files into a single output and removes temporary files.""" + """ Function that concatenates temporary files into a single output and removes temporary files. """ src_path = Path(kwargs['path'], '*.bin.tmp') dest_path = Path(kwargs['path'], 'data.bin') From 9513534d2f80c99fc4c239d6070804150e282ac0 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Sun, 9 May 2021 02:44:49 +0200 Subject: [PATCH 02/21] CG motif analysis --- analysis/results/CG_mod.csv | 1001 +++++++++++++++++++++++++++++++++ analysis/results/CG_nomod.csv | 1001 +++++++++++++++++++++++++++++++++ 2 files changed, 2002 insertions(+) create mode 100644 analysis/results/CG_mod.csv create mode 100644 analysis/results/CG_nomod.csv diff --git a/analysis/results/CG_mod.csv b/analysis/results/CG_mod.csv new file mode 100644 index 0000000..fd19206 --- /dev/null +++ b/analysis/results/CG_mod.csv @@ -0,0 +1,1001 @@ +read_id,alignment_len,M,X,D,I,M_CG,X_CG,D_CG,I_CG,mapq +00417c91-cb24-4bf6-99e5-6fed9d6cfab3,4038,3436,221,238,143,211,21,8,2,60 +005c0804-25d2-47eb-9118-fca1ee7b19ef,2219,2099,26,84,10,139,4,3,0,60 +008b7b57-76f8-4762-a467-56f6852da36d,953,867,29,30,27,54,2,0,1,60 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+fcb61942-bc95-456d-aede-a22baa242c58,0,0,0,0,0,0,0,0,0,0 +fccb7c07-ee14-478a-8503-f108ccbb163d,640,608,11,5,16,55,3,1,1,60 +fce40a5a-b7dc-42b2-aa13-15fe0bfe8bf2,276,270,0,6,0,13,0,0,0,60 +fd7f5f8d-6c7a-4d19-9455-a3bdef0b00ef,5823,5469,104,181,69,502,2,1,3,60 +fd877052-ebdd-406d-8604-1212f76c1e2e,584,551,10,20,3,49,3,0,0,60 +fde5ec84-1d26-462d-be93-5d68b7b08c68,2021,1888,41,69,23,162,5,2,0,60 +fe44fc31-e09c-4d65-8705-cf3fd81bb3e2,909,879,13,4,13,35,0,0,0,60 +fe70e8a7-0b33-426c-a13b-3ce3969cd0f4,0,0,0,0,0,0,0,0,0,0 +ff998c2f-716c-4447-911d-e9f51f9a8316,1275,1182,38,28,27,61,5,0,2,60 From 242f889fbcf65d5eac52563a89d9bfb998d48512 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Tue, 11 May 2021 23:26:11 +0200 Subject: [PATCH 03/21] Added code for CG motif analysis --- analysis/scripts/analyse_CG.py | 169 +++++++++++++++++++++++++++++++++ 1 file changed, 169 insertions(+) create mode 100644 analysis/scripts/analyse_CG.py diff --git a/analysis/scripts/analyse_CG.py b/analysis/scripts/analyse_CG.py new file mode 100644 index 0000000..96f1d91 --- /dev/null +++ b/analysis/scripts/analyse_CG.py @@ -0,0 +1,169 @@ +from pathlib import Path +import mappy as mp +from tqdm import tqdm +import h5py +import io +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord +import pandas as pd + + +def get_files(path, recursive=False): + if path.is_file(): + return {path} + + # Finding all input FAST5 files + if recursive: + files = path.glob('**/*.fast5') + else: + files = path.glob('*.fast5') + + return set(files) + + +def read_fastq(read) -> SeqRecord: + fastq_str = read[f'/Analyses/Basecall_1D_000/BaseCalled_template/Fastq'][()] + fastq_str = fastq_str.decode() + str_file = io.StringIO(fastq_str) + + record = SeqIO.read(str_file, 'fastq') + return record + + +def count_matches(cs): + matches = {'M': 0, 'X': 0, 'D': 0, 'I': 0} + mode, value = 'M', 0 + + for c in cs: + if c == ':': + matches[mode] += int(value) if mode == 'M' or mode == 'X' else len(value) + mode, value = 'M', '' + continue + elif c == '*': + matches[mode] += int(value) if mode == 'M' or mode == 'X' else len(value) + mode, value = 'X', '' + continue + elif c == '-': + matches[mode] += int(value) if mode == 'M' or mode == 'X' else len(value) + mode, value = 'D', '' + continue + elif c == '+': + matches[mode] += int(value) if mode == 'M' or mode == 'X' else len(value) + mode, value = 'I', '' + continue + + if mode == 'X': + value = 1 + else: + value += c + + matches[mode] += int(value) if mode == 'M' or mode == 'X' else len(value) + return matches + + +def normalize(ref, query, cigar): + r = list(ref) + q = list(query) + i = 0 + num = '' + + for c in cigar: + if c.isnumeric(): + num += c + continue + + elif c == 'M': + i += int(num) + + elif c == 'D': + for _ in range(int(num)): + q.insert(i, '-') + i += 1 + + elif c == 'I': + for _ in range(int(num)): + r.insert(i, '-') + i += 1 + + num = '' + + return ''.join(r), ''.join(q) + + +def count_CG(ref, query): + CG_cnt = {'M': 0, 'X': 0, 'D': 0, 'I': 0} + + for i in range(len(ref) - 1): + if ref[i] == 'C' and ref[i + 1] == 'G': + if query[i] == 'C' and query[i + 1] == 'G': + CG_cnt['M'] += 1 + + elif query[i] == '-' and query[i + 1] == 'G': + CG_cnt['D'] += 1 + + elif query[i] in {'A', 'G', 'T'} and query[i + 1] == 'G': + CG_cnt['X'] += 1 + + elif ref[i] == '-' and ref[i + 1] == 'G': + if query[i] == 'C' and query[i + 1] == 'G': + CG_cnt['I'] += 1 + + return CG_cnt + + +def analyse_CG(dir_in, file_out, file_fasta): + a = mp.Aligner(file_fasta) # Load or build index + if not a: + raise Exception("ERROR: failed to load/build index") + + reads = get_files(dir_in) + data = [] + + for read in tqdm(reads): + with h5py.File(read, 'r', libver='latest') as fd: + matches = {'M': 0, 'X': 0, 'D': 0, 'I': 0} + CG_cnt = {'M': 0, 'X': 0, 'D': 0, 'I': 0} + + fastq = read_fastq(fd) + ref = '' + mapq = 0 + + for hit in a.map(fastq.seq, cs=True): # Traverse alignments + if hit.is_primary: # Check if the alignment is primary + # Alignment + matches = count_matches(hit.cs) + + # Reference + for seq_record in SeqIO.parse(file_fasta, 'fasta'): + ref = seq_record.seq[hit.r_st: hit.r_en] + + # Query + query = fastq.seq[hit.q_st: hit.q_en] + if hit.strand == -1: + query = mp.revcomp(query) + + # Normalize + ref, query = normalize(ref, query, hit.cigar_str) + + # Analyse CG motif + CG_cnt = count_CG(ref, query) + + mapq = hit.mapq + break + + data.append([fastq.id, len(ref), matches['M'], matches['X'], matches['D'], matches['I'], + CG_cnt['M'], CG_cnt['X'], CG_cnt['D'], CG_cnt['I'], mapq]) + + data = pd.DataFrame(data, columns=['read_id', 'alignment_len', 'M', 'X', 'D', 'I', + 'M_CG', 'X_CG', 'D_CG', 'I_CG', 'mapq']) + data.sort_values('read_id', inplace=True) + data.to_csv(file_out, index=False) + + +if __name__ == '__main__': + modification = 'nomod' + dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') + file_out = f'/home/sdeur/tmp-mappy/CG_{modification}.csv' + file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' + + analyse_CG(dir_in, file_out, file_fasta) From 7f873a03bdc006cd79b5b74a557fe07cdfe2963f Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 12 May 2021 00:15:13 +0200 Subject: [PATCH 04/21] Analysis of mappy output --- analysis/results/mappy_mod.csv | 1001 ++++++++++++++++++++++++++++++ analysis/results/mappy_nomod.csv | 1001 ++++++++++++++++++++++++++++++ analysis/scripts/align_mappy.py | 82 +++ 3 files changed, 2084 insertions(+) create mode 100644 analysis/results/mappy_mod.csv create mode 100644 analysis/results/mappy_nomod.csv create mode 100644 analysis/scripts/align_mappy.py diff --git a/analysis/results/mappy_mod.csv b/analysis/results/mappy_mod.csv new file mode 100644 index 0000000..86c6945 --- /dev/null +++ b/analysis/results/mappy_mod.csv @@ -0,0 +1,1001 @@ +read_id,r_st,r_en,q_st,q_en,r_CG_num,q_CG_num,cigar_str 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+import h5py +import io +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord +import re +import pandas as pd + + +def get_files(path, recursive=False): + if path.is_file(): + return {path} + + # Finding all input FAST5 files + if recursive: + files = path.glob('**/*.fast5') + else: + files = path.glob('*.fast5') + + return set(files) + + +def read_fastq(read) -> SeqRecord: + fastq_str = read[f'/Analyses/Basecall_1D_000/BaseCalled_template/Fastq'][()] + fastq_str = fastq_str.decode() + str_file = io.StringIO(fastq_str) + + record = SeqIO.read(str_file, 'fastq') + return record + + +def align_mappy(dir_in, file_out, file_fasta): + a = mp.Aligner(file_fasta) # Load or build index + if not a: + raise Exception("ERROR: failed to load/build index") + + reads = get_files(dir_in) + files_fastq = {} + data = [] + + for read in tqdm(reads): + with h5py.File(read, 'r', libver='latest') as fd: + no_alignment = True + fastq = read_fastq(fd) + files_fastq[fastq.id] = len(fastq.seq) + + for hit in a.map(fastq.seq): # Traverse alignments + if hit.is_primary: # Check if the alignment is primary + # Reference + for seq_record in SeqIO.parse(file_fasta, 'fasta'): + ref = seq_record.seq[hit.r_st: hit.r_en] + r_CG_num = len(re.findall(r'(CG)', str(ref))) + + # Query + query = fastq.seq[hit.q_st: hit.q_en] + if hit.strand == -1: + query = mp.revcomp(query) + q_CG_num = len(re.findall(r'(CG)', str(query))) + + no_alignment = False + data.append([fastq.id, hit.r_st, hit.r_en, hit.q_st, hit.q_en, r_CG_num, q_CG_num, hit.cigar_str]) + break + + if no_alignment: + data.append([fastq.id, '', '', '', '', 0, 0, '']) + + data = pd.DataFrame(data, columns=['read_id', 'r_st', 'r_en', 'q_st', 'q_en', 'r_CG_num', 'q_CG_num', 'cigar_str']) + data.sort_values('read_id', inplace=True) + data.to_csv(file_out, index=False) + + print("Average length of fastq files:", sum(files_fastq.values()) / len(files_fastq.values())) + + +if __name__ == '__main__': + modification = 'nomod' + dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') + file_out = f'/home/sdeur/tmp-mappy/mappy_{modification}.csv' + file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' + + align_mappy(dir_in, file_out, file_fasta) From 425cfcc8581198567040b330f1d0b6d646d0983c Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 12 May 2021 00:33:06 +0200 Subject: [PATCH 05/21] Plot mapping quality --- analysis/plots/mapq_mod.png | Bin 0 -> 20419 bytes analysis/plots/mapq_nomod.png | Bin 0 -> 20993 bytes analysis/scripts/align_mappy.py | 24 +-------------- analysis/scripts/analyse_CG.py | 24 +-------------- analysis/scripts/plot_mapq.py | 50 ++++++++++++++++++++++++++++++++ 5 files changed, 52 insertions(+), 46 deletions(-) create mode 100644 analysis/plots/mapq_mod.png create mode 100644 analysis/plots/mapq_nomod.png create mode 100644 analysis/scripts/plot_mapq.py diff --git a/analysis/plots/mapq_mod.png b/analysis/plots/mapq_mod.png new file mode 100644 index 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zwkSzZ>X7MOwtzl7jY?UC%vkgM>;~fOjE;`}G*ow7^5VrhybU58K{q;igtA3_1={Q@ mbb>cg(*K<|6qYWpD*1d@%KPhj@M;ta_3Xtn2|r)E_5T4BGgGwy literal 0 HcmV?d00001 diff --git a/analysis/scripts/align_mappy.py b/analysis/scripts/align_mappy.py index a38fa26..e2bc7e1 100644 --- a/analysis/scripts/align_mappy.py +++ b/analysis/scripts/align_mappy.py @@ -2,33 +2,11 @@ import mappy as mp from tqdm import tqdm import h5py -import io from Bio import SeqIO -from Bio.SeqRecord import SeqRecord import re import pandas as pd - -def get_files(path, recursive=False): - if path.is_file(): - return {path} - - # Finding all input FAST5 files - if recursive: - files = path.glob('**/*.fast5') - else: - files = path.glob('*.fast5') - - return set(files) - - -def read_fastq(read) -> SeqRecord: - fastq_str = read[f'/Analyses/Basecall_1D_000/BaseCalled_template/Fastq'][()] - fastq_str = fastq_str.decode() - str_file = io.StringIO(fastq_str) - - record = SeqIO.read(str_file, 'fastq') - return record +from util import get_files, read_fastq def align_mappy(dir_in, file_out, file_fasta): diff --git a/analysis/scripts/analyse_CG.py b/analysis/scripts/analyse_CG.py index 96f1d91..a42d5dd 100644 --- a/analysis/scripts/analyse_CG.py +++ b/analysis/scripts/analyse_CG.py @@ -2,32 +2,10 @@ import mappy as mp from tqdm import tqdm import h5py -import io from Bio import SeqIO -from Bio.SeqRecord import SeqRecord import pandas as pd - -def get_files(path, recursive=False): - if path.is_file(): - return {path} - - # Finding all input FAST5 files - if recursive: - files = path.glob('**/*.fast5') - else: - files = path.glob('*.fast5') - - return set(files) - - -def read_fastq(read) -> SeqRecord: - fastq_str = read[f'/Analyses/Basecall_1D_000/BaseCalled_template/Fastq'][()] - fastq_str = fastq_str.decode() - str_file = io.StringIO(fastq_str) - - record = SeqIO.read(str_file, 'fastq') - return record +from util import get_files, read_fastq def count_matches(cs): diff --git a/analysis/scripts/plot_mapq.py b/analysis/scripts/plot_mapq.py new file mode 100644 index 0000000..f4330e9 --- /dev/null +++ b/analysis/scripts/plot_mapq.py @@ -0,0 +1,50 @@ +from pathlib import Path +import mappy as mp +from tqdm import tqdm +import h5py +import matplotlib.pyplot as plt +import numpy as np + +from util import get_files, read_fastq + + +def plot_distribution(data, modification, bins=15): + """ Distribution of mapping quality """ + plt.hist(data, bins=bins, color='green' if modification == 'nomod' else 'red') + plt.title(f'Distribution of mapping quality ({modification})') + plt.xlabel('mapping quality') + plt.ylabel('count') + plt.yticks(np.arange(0, 1001, 100)) + plt.savefig(f'../plots/mapq_{modification}.png') + + +def get_mapqs(dir_in, file_fasta): + a = mp.Aligner(file_fasta) # Load or build index + if not a: + raise Exception("ERROR: failed to load/build index") + + reads = get_files(dir_in) + mapqs = [] + + for read in tqdm(reads): + with h5py.File(read, 'r', libver='latest') as fd: + fastq = read_fastq(fd) + mapq = 0 + + for hit in a.map(fastq.seq): # Traverse alignments + if hit.is_primary: # Check if the alignment is primary + mapq = hit.mapq + break + + mapqs.append(mapq) + + return mapqs + + +if __name__ == '__main__': + modification = 'nomod' + dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') + file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' + + mapqs = get_mapqs(dir_in, file_fasta) + plot_distribution(mapqs, modification) From 91a1089f68241092be16155a948f658ab958f0d9 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 12 May 2021 01:49:13 +0200 Subject: [PATCH 06/21] Plot start raw index --- analysis/plots/start_raw_index_mod.png | Bin 0 -> 17173 bytes analysis/plots/start_raw_index_nomod.png | Bin 0 -> 17425 bytes analysis/scripts/align_mappy.py | 2 +- analysis/scripts/analyse_CG.py | 2 +- analysis/scripts/plot_mapq.py | 2 +- analysis/scripts/plot_start_raw_index.py | 149 +++++++++++++++++++++++ analysis/scripts/util.py | 25 ++++ 7 files 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oE`Ldu%Eh?+|Kk6mqGEZK=bhqfcWWMI;Y}22Df!bWCokRl9|F$S!~g&Q literal 0 HcmV?d00001 diff --git a/analysis/scripts/align_mappy.py b/analysis/scripts/align_mappy.py index e2bc7e1..ab28b45 100644 --- a/analysis/scripts/align_mappy.py +++ b/analysis/scripts/align_mappy.py @@ -52,7 +52,7 @@ def align_mappy(dir_in, file_out, file_fasta): if __name__ == '__main__': - modification = 'nomod' + modification = 'nomod' # 'mod' or 'nomod' dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') file_out = f'/home/sdeur/tmp-mappy/mappy_{modification}.csv' file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' diff --git a/analysis/scripts/analyse_CG.py b/analysis/scripts/analyse_CG.py index a42d5dd..f21d5df 100644 --- a/analysis/scripts/analyse_CG.py +++ b/analysis/scripts/analyse_CG.py @@ -139,7 +139,7 @@ def analyse_CG(dir_in, file_out, file_fasta): if __name__ == '__main__': - modification = 'nomod' + modification = 'nomod' # 'mod' or 'nomod' dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') file_out = f'/home/sdeur/tmp-mappy/CG_{modification}.csv' file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' diff --git a/analysis/scripts/plot_mapq.py b/analysis/scripts/plot_mapq.py index f4330e9..4e541fd 100644 --- a/analysis/scripts/plot_mapq.py +++ b/analysis/scripts/plot_mapq.py @@ -42,7 +42,7 @@ def get_mapqs(dir_in, file_fasta): if __name__ == '__main__': - modification = 'nomod' + modification = 'nomod' # 'mod' or 'nomod' dir_in = Path(f'/home/sdeur/tmp-guppy/{modification}/workspace') file_fasta = '/home/sdeur/data/ecoli_k12_mg1655.fasta' diff --git a/analysis/scripts/plot_start_raw_index.py b/analysis/scripts/plot_start_raw_index.py new file mode 100644 index 0000000..ce3fc32 --- /dev/null +++ b/analysis/scripts/plot_start_raw_index.py @@ -0,0 +1,149 @@ +import os +import numpy as np +from ont_fast5_api.fast5_interface import get_fast5_file +import matplotlib.pyplot as plt +import statistics + + +def get_raw_signal_block(fast5_filename, read_id, start_seq_position, end_seq_position): + raw_data = _get_raw_data(fast5_filename, read_id) + (move_table, first_sample_template, block_stride) = _get_fast5_data(fast5_filename, read_id) + seq_to_move_map = _create_map_from_basecall_to_signal(move_table) + _, start_raw_index = _get_raw_signal_block(raw_data, seq_to_move_map, first_sample_template, block_stride, + start_seq_position, end_seq_position) + return list(raw_data), start_raw_index + + +def _get_raw_signal_block(raw_data, seq_to_move_map, first_sample_template, block_stride, + start_seq_position, end_seq_position): + start_raw_index = first_sample_template + seq_to_move_map[start_seq_position] * block_stride + # Get the raw index of the *next* entry in the seq_to_move map, + # so that we include all the raw data for end_seq_position + if end_seq_position + 1 == len(seq_to_move_map): + end_raw_index = len(raw_data) + else: + end_raw_index = first_sample_template + seq_to_move_map[end_seq_position + 1] * block_stride + return raw_data[start_raw_index:end_raw_index], start_raw_index + + +def _get_raw_data(fast5_filename, read_id): + with get_fast5_file(fast5_filename, mode="r") as f5file: + read = f5file.get_read(read_id) + raw_data = read.get_raw_data() + return raw_data + + +def _create_map_from_basecall_to_signal(move_table): + cumulative_moves = np.add.accumulate(move_table) + sequence_to_move_index = [np.min(np.where(cumulative_moves == x)) + for x in + range(1, int(np.max(cumulative_moves) + 1))] + return sequence_to_move_index + + +def _get_fast5_data(fast5_filename, read_id): + with get_fast5_file(fast5_filename) as f5file: + read = f5file.get_read(read_id) + basecall_analysis = read.get_latest_analysis('Basecall_1D') + segmentation_analysis = read.get_latest_analysis('Segmentation') + segmentation_summary = read.get_summary_data(segmentation_analysis) + basecall_summary = read.get_summary_data(basecall_analysis) + move_table = read.get_analysis_dataset(basecall_analysis, 'BaseCalled_template/Move') + first_sample_template = segmentation_summary['segmentation']['first_sample_template'] + block_stride = basecall_summary['basecall_1d_template']['block_stride'] + return move_table, first_sample_template, block_stride + + +def plot_distribution(dir_in, files_fastq, bins=50): + """ Distribution of start_raw_index """ + data = [] + + for file in os.listdir(dir_in): + fast5_filename = os.path.join(dir_in, file) + read_id = file[:-6] + start_seq_position = 0 + end_seq_position = files_fastq[read_id] - 1 + _, start_raw_index = get_raw_signal_block(fast5_filename, read_id, start_seq_position, end_seq_position) + data.append(start_raw_index) + + plt.hist(data, bins=bins, color='green' if modification == 'nomod' else 'red') + plt.title(f'Distribution of "start_raw_index" ({modification})') + plt.xlabel('start_raw_index') + plt.ylabel('count') + plt.xticks(np.arange(0, 5001, 500)) # Change if needed + plt.yticks(np.arange(0, 151, 25)) # Change if needed + plt.savefig(f'../plots/start_raw_index_{modification}.png') + + +def plot_raw_signal(dir_in, files_fastq, fast5_file, n=1000): + """ raw signal data + start_raw_index line """ + fast5_filename = os.path.join(dir_in, fast5_file) + read_id = fast5_file[:-6] + start_seq_position = 0 + end_seq_position = files_fastq[read_id] - 1 + raw_data, start_raw_index = get_raw_signal_block(fast5_filename, read_id, start_seq_position, end_seq_position) + + x_first_n = [str(x) for x in range(0, n)] + x_last_n = [str(x) for x in range(len(raw_data) - n, len(raw_data))] + y_first_n = raw_data[:n] + y_last_n = raw_data[-n:] + + x = x_first_n + x_last_n + y = y_first_n + y_last_n + + fig, ax = plt.subplots(1, 1) + ax.plot(x, y, color='green' if modification == 'nomod' else 'red') + plt.axvline(x=str(start_raw_index), color='darkblue', linestyle='--', label=f'start_raw_index = {start_raw_index}') + + ax.set_xticks(x[::100]) # Change if needed + ax.set_xticklabels(x[::100], rotation=45) # Change if needed + plt.title(f'read_id: {read_id}') + plt.xlabel('time') + plt.ylabel('raw signal data') + plt.legend() + plt.savefig(f'../plots/raw_signal_{read_id}.png') + + +def plot_stdev(dir_in, files_fastq, fast5_file, n=1000, window=50, step=5): + """ Standard deviation of the raw signal """ + fast5_filename = os.path.join(dir_in, fast5_file) + read_id = fast5_file[:-6] + start_seq_position = 0 + end_seq_position = files_fastq[read_id] - 1 + raw_data, start_raw_index = get_raw_signal_block(fast5_filename, read_id, start_seq_position, end_seq_position) + + first_n = raw_data[:n] + last_n = raw_data[-n:] + + stdev_first_n = [] + stdev_last_n = [] + for i in range(0, n - window, step): + points_first_n = np.array(first_n[i: i + window], dtype=np.float64) + stdev_first_n.append(statistics.stdev(points_first_n)) + points_last_n = np.array(last_n[i: i + window], dtype=np.float64) + stdev_last_n.append(statistics.stdev(points_last_n)) + + fig, axs = plt.subplots(2) + fig.suptitle(f'Standard deviation - {read_id}') + axs[0].plot(range(0, n - window, step), stdev_first_n, color='green' if modification == 'nomod' else 'red') + axs[1].plot(range(0, n - window, step), stdev_last_n, color='green' if modification == 'nomod' else 'red') + plt.xlabel('time') + axs[0].set_ylabel(f'stdev - first {n}') + axs[1].set_ylabel(f'stdev - last {n}') + plt.savefig(f'../plots/stdev_{read_id}.png') + + +if __name__ == '__main__': + modification = 'nomod' # 'mod' or 'nomod' + dir_in_fastq = f'/home/sdeur/tmp-guppy/{modification}-fastq' + dir_in = f'/home/sdeur/tmp-guppy/{modification}/workspace' + + files_fastq = {} # {read_id : fastq_len} + for file_fastq in os.listdir(dir_in_fastq): + with open(os.path.join(dir_in_fastq, file_fastq)) as fin: + files_fastq[file_fastq[:-6]] = len(fin.readlines()[1].strip()) + + # Choose between the following plotting methods + plot_distribution(dir_in, files_fastq) + plot_raw_signal(dir_in, files_fastq, 'a40f1242-df43-4b9d-a97e-d9af64f3c956.fast5') + plot_stdev(dir_in, files_fastq, 'a40f1242-df43-4b9d-a97e-d9af64f3c956.fast5') diff --git a/analysis/scripts/util.py b/analysis/scripts/util.py new file mode 100644 index 0000000..9397302 --- /dev/null +++ b/analysis/scripts/util.py @@ -0,0 +1,25 @@ +import io +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord + + +def get_files(path, recursive=False): + if path.is_file(): + return {path} + + # Finding all input FAST5 files + if recursive: + files = path.glob('**/*.fast5') + else: + files = path.glob('*.fast5') + + return set(files) + + +def read_fastq(read) -> SeqRecord: + fastq_str = read[f'/Analyses/Basecall_1D_000/BaseCalled_template/Fastq'][()] + fastq_str = fastq_str.decode() + str_file = io.StringIO(fastq_str) + + record = SeqIO.read(str_file, 'fastq') + return record From 0bc7ad487a1a0ab39abce02fea0cdc820ac47d5d Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 12 May 2021 01:54:15 +0200 Subject: [PATCH 07/21] Plot raw 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a = mp.Aligner(file_fasta) # Load or build index + a = mp.Aligner(file_fasta, preset='map-ont') # Load or build index if not a: raise Exception("ERROR: failed to load/build index") diff --git a/analysis/scripts/analyse_CG.py b/analysis/scripts/analyse_CG.py index f21d5df..9efa362 100644 --- a/analysis/scripts/analyse_CG.py +++ b/analysis/scripts/analyse_CG.py @@ -90,7 +90,7 @@ def count_CG(ref, query): def analyse_CG(dir_in, file_out, file_fasta): - a = mp.Aligner(file_fasta) # Load or build index + a = mp.Aligner(file_fasta, preset='map-ont') # Load or build index if not a: raise Exception("ERROR: failed to load/build index") diff --git a/analysis/scripts/plot_mapq.py b/analysis/scripts/plot_mapq.py index 4e541fd..3efe635 100644 --- a/analysis/scripts/plot_mapq.py +++ b/analysis/scripts/plot_mapq.py @@ -19,7 +19,7 @@ def plot_distribution(data, modification, bins=15): def get_mapqs(dir_in, file_fasta): - a = mp.Aligner(file_fasta) # Load or build index + a = mp.Aligner(file_fasta, preset='map-ont') # Load or build index if not a: raise Exception("ERROR: failed to load/build index") diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py new file mode 100644 index 0000000..188d540 --- /dev/null +++ b/remove-tombo/custom_processor.py @@ -0,0 +1,170 @@ +from pathlib import Path +from tqdm import tqdm +from pyguppyclient import GuppyBasecallerClient, get_fast5_files, yield_reads +import mappy as mp +from Bio import SeqIO +import re +import numpy as np + + +# config='dna_r9.4.1_450bps_hac.cfg' for more precise basecalling +def basecall(read_file, config='dna_r9.4.1_450bps_fast'): + with GuppyBasecallerClient(config_name=config, trace=True) as client: + for read in yield_reads(read_file): + called = client.basecall(read)[1] + return read, called + + +def align(reference_file, query, mapq): + a = mp.Aligner(reference_file, preset='map-ont') # Load or build index + if not a: + raise Exception("ERROR: failed to load/build index") + + for hit in a.map(query): # Traverse alignments + if hit.is_primary: # Check if the alignment is primary + if hit.mapq < mapq: # Check if the mapping quality is below set threshold + return None + return hit + + return None + + +def get_motif_positions(reference_file, alignment, motif, index): + for seq_record in SeqIO.parse(reference_file, 'fasta'): + reference = str(seq_record.seq[alignment.r_st: alignment.r_en]) + + if alignment.strand == -1: # Reverse alignment strand + reference = mp.revcomp(reference) + + return [m.start() + index for m in re.finditer(motif, reference, re.I)] + + +def sequence_to_raw(read, called): + first_signal_id = len(read.signal) - called.trimmed_samples + move_index = np.nonzero(called.move)[0] + + seq_to_raw_start = first_signal_id + move_index * called.model_stride + seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) + seq_to_raw_end = seq_to_raw_start + seq_to_raw_len + + return list(zip(seq_to_raw_start, seq_to_raw_end)) + + +def reference_to_basecall(alignment): + cigar = alignment.cigar if alignment.strand == 1 else reversed(alignment.cigar) + + ref_to_bc = [] + qpos = alignment.q_st + + for length, operation in cigar: + if operation in {0, 7, 8}: # Match or mismatch + for i in range(length): + ref_to_bc.append((qpos + i, qpos + i + 1)) + qpos += length + + elif operation == 1: # Insertion + qpos += length + + elif operation in {2, 3}: # Deletion or skip + for _ in range(length): + ref_to_bc.append((qpos, qpos)) + + return ref_to_bc + + +def resolve_insertions(position, ref_to_bc, seq_to_raw, window): + base_intervals = ref_to_bc[position - window: position + window + 1] # End is exclusive + signal_intervals = seq_to_raw[base_intervals[0][0]: base_intervals[-1][1]] + + deletion_idx = [] + + for i in range(len(base_intervals)): + if base_intervals[i][0] == base_intervals[i][1]: # Deletion or skip + signal_intervals.insert(i, (seq_to_raw[base_intervals[i][0]][0], seq_to_raw[base_intervals[i][0]][0])) + + if deletion_idx and i == deletion_idx[-1][1]: + deletion_idx[-1] = deletion_idx[-1][0], i + 1 + else: + deletion_idx.append((i, i + 1)) + + if i != len(base_intervals) - 1 and base_intervals[i][1] != base_intervals[i + 1][0]: # Resolve insertions + no_of_insertions = base_intervals[i + 1][0] - base_intervals[i][1] + insertion_interval = signal_intervals[i][1], signal_intervals[i + 1 + no_of_insertions][0] + + center = int(np.mean(insertion_interval)) + del signal_intervals[i + 1: i + 1 + no_of_insertions] + + signal_intervals[i] = signal_intervals[i][0], center # Base to the left + signal_intervals[i + 1] = center, signal_intervals[i + 1][1] # Base to the right + + return base_intervals, signal_intervals, deletion_idx + + +def resolve_deletions(signal_intervals, deletion_idx): + for idx_st, idx_en in deletion_idx: # Resolve deletions + if idx_st == 0 and idx_en == len(signal_intervals): # Region consisting only of deletions + break + + if idx_st == 0: + start, end = idx_st, idx_en + elif idx_en == len(signal_intervals): + start, end = idx_st - 1, idx_en - 1 + else: + start, end = idx_st - 1, idx_en + + sig_st, sig_en = signal_intervals[start][0], signal_intervals[end][1] + intervals = np.array_split(range(sig_st, sig_en), end - start + 1) + + for i in range(start, end + 1): + interval = intervals.pop(0) + if len(interval) == 0: # If there is not enough signal points for the remaining bases + break + signal_intervals[i] = interval[0], interval[-1] + 1 + + return signal_intervals + + +def resolve_region(position, ref_to_bc, seq_to_raw, window): + if position - 8 < 0 or position + 8 >= len(ref_to_bc): + return None + + base_intervals, signal_intervals, deletion_idx = resolve_insertions(position, ref_to_bc, seq_to_raw, window) + signal_intervals = resolve_deletions(signal_intervals, deletion_idx) + + assert len(base_intervals) == len(signal_intervals) + + return signal_intervals + + +def custom_processor(fast5_file, reference_file, mapq=0, motif='CG', index=0, window=8): + read, called = basecall(fast5_file) + + alignment = align(reference_file, called.seq, mapq) + if not alignment: + return + + motif_positions = get_motif_positions(reference_file, alignment, motif, index) + + ref_to_bc = reference_to_basecall(alignment) + seq_to_raw = sequence_to_raw(read, called) + + for motif_position in motif_positions: + event_intervals = resolve_region(motif_position, ref_to_bc, seq_to_raw, window) + if event_intervals is None: + continue + + position = alignment.r_st + motif_position + event_lens = [end - start for start, end in event_intervals] + + print(f'position: {position}\nevent_intervals: {event_intervals}\nevent_lens:{event_lens}\n') + + +if __name__ == '__main__': + modification = 'mod' # 'mod' or 'nomod' + dir_in = Path(f'/home/sdeur/data/{modification}') + reference_file = '/home/sdeur/data/ecoli_k12_mg1655.fasta' + + fast5_files = get_fast5_files(dir_in, recursive=True) + + for fast5_file in tqdm(fast5_files): + custom_processor(fast5_file, reference_file) From 0c3690a4cf260a8620e855b98089a91d7e144c5b Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Sun, 30 May 2021 01:37:24 +0200 Subject: [PATCH 09/21] Improve custom processor --- remove-tombo/custom_processor.py | 109 +++++++++++++++++++++---------- src/extract_features.py | 9 +-- 2 files changed, 77 insertions(+), 41 deletions(-) diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index 188d540..1f14c8c 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -6,8 +6,39 @@ import re import numpy as np +from util import ResegmentationData -# config='dna_r9.4.1_450bps_hac.cfg' for more precise basecalling + +def make_aligner(reference_file): + aligner = mp.Aligner(reference_file, preset='map-ont') # Load or build index + if not aligner: + raise Exception("ERROR: failed to load/build index") + + return aligner + + +def get_reference(reference_file): + for seq_record in SeqIO.parse(reference_file, 'fasta'): + reference = str(seq_record.seq) + + return reference + + +def get_motif_positions(reference, motif, index): + r_len = len(reference) + + # Forward strand + fwd_matches = re.finditer(motif, reference, re.I) + fwd_pos = set(m.start() + index for m in fwd_matches) + + # Reverse strand + rev_matches = re.finditer(motif, mp.revcomp(reference), re.I) + rev_pos = set(r_len - (m.start() + index) - 1 for m in rev_matches) + + return fwd_pos, rev_pos + + +# config='dna_r9.4.1_450bps_hac' for more precise basecalling def basecall(read_file, config='dna_r9.4.1_450bps_fast'): with GuppyBasecallerClient(config_name=config, trace=True) as client: for read in yield_reads(read_file): @@ -15,12 +46,8 @@ def basecall(read_file, config='dna_r9.4.1_450bps_fast'): return read, called -def align(reference_file, query, mapq): - a = mp.Aligner(reference_file, preset='map-ont') # Load or build index - if not a: - raise Exception("ERROR: failed to load/build index") - - for hit in a.map(query): # Traverse alignments +def align(aligner, query, mapq): + for hit in aligner.map(query): # Traverse alignments if hit.is_primary: # Check if the alignment is primary if hit.mapq < mapq: # Check if the mapping quality is below set threshold return None @@ -29,25 +56,15 @@ def align(reference_file, query, mapq): return None -def get_motif_positions(reference_file, alignment, motif, index): - for seq_record in SeqIO.parse(reference_file, 'fasta'): - reference = str(seq_record.seq[alignment.r_st: alignment.r_en]) +def get_relevant_motif_positions(motif_positions, alignment): + strand_pos = motif_positions[0] if alignment.strand == 1 else motif_positions[1] - if alignment.strand == -1: # Reverse alignment strand - reference = mp.revcomp(reference) + relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) - return [m.start() + index for m in re.finditer(motif, reference, re.I)] - - -def sequence_to_raw(read, called): - first_signal_id = len(read.signal) - called.trimmed_samples - move_index = np.nonzero(called.move)[0] - - seq_to_raw_start = first_signal_id + move_index * called.model_stride - seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) - seq_to_raw_end = seq_to_raw_start + seq_to_raw_len - - return list(zip(seq_to_raw_start, seq_to_raw_end)) + if alignment.strand == 1: + return {p - alignment.r_st for p in relevant_positions} + else: + return {alignment.r_en - 1 - p for p in relevant_positions} def reference_to_basecall(alignment): @@ -72,6 +89,17 @@ def reference_to_basecall(alignment): return ref_to_bc +def sequence_to_raw(read, called): + first_signal_id = len(read.signal) - called.trimmed_samples + move_index = np.nonzero(called.move)[0] + + seq_to_raw_start = first_signal_id + move_index * called.model_stride + seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) + seq_to_raw_end = seq_to_raw_start + seq_to_raw_len + + return list(zip(seq_to_raw_start, seq_to_raw_end)) + + def resolve_insertions(position, ref_to_bc, seq_to_raw, window): base_intervals = ref_to_bc[position - window: position + window + 1] # End is exclusive signal_intervals = seq_to_raw[base_intervals[0][0]: base_intervals[-1][1]] @@ -136,27 +164,43 @@ def resolve_region(position, ref_to_bc, seq_to_raw, window): return signal_intervals -def custom_processor(fast5_file, reference_file, mapq=0, motif='CG', index=0, window=8): +def custom_processor(fast5_file, aligner, reference, motif_positions, mapq, window): read, called = basecall(fast5_file) - alignment = align(reference_file, called.seq, mapq) + alignment = align(aligner, called.seq, mapq) if not alignment: - return + return None - motif_positions = get_motif_positions(reference_file, alignment, motif, index) + relevant_motif_positions = get_relevant_motif_positions(motif_positions, alignment) ref_to_bc = reference_to_basecall(alignment) seq_to_raw = sequence_to_raw(read, called) - for motif_position in motif_positions: + for motif_position in relevant_motif_positions: event_intervals = resolve_region(motif_position, ref_to_bc, seq_to_raw, window) if event_intervals is None: continue position = alignment.r_st + motif_position event_lens = [end - start for start, end in event_intervals] + bases = reference[position - window: position + window + 1] + + return ResegmentationData(position, event_intervals, event_lens, bases) + + +def process_data(dir_in, reference_file, mapq=0, motif='CG', index=0, window=8): + fast5_files = get_fast5_files(dir_in, recursive=True) + + aligner = make_aligner(reference_file) + reference = get_reference(reference_file) + motif_positions = get_motif_positions(reference, motif, index) - print(f'position: {position}\nevent_intervals: {event_intervals}\nevent_lens:{event_lens}\n') + for fast5_file in tqdm(fast5_files): + resegmentation_data = custom_processor(fast5_file, aligner, reference, motif_positions, mapq, window) + if resegmentation_data is None: + continue + + print(resegmentation_data) if __name__ == '__main__': @@ -164,7 +208,4 @@ def custom_processor(fast5_file, reference_file, mapq=0, motif='CG', index=0, wi dir_in = Path(f'/home/sdeur/data/{modification}') reference_file = '/home/sdeur/data/ecoli_k12_mg1655.fasta' - fast5_files = get_fast5_files(dir_in, recursive=True) - - for fast5_file in tqdm(fast5_files): - custom_processor(fast5_file, reference_file) + process_data(dir_in, reference_file) diff --git a/src/extract_features.py b/src/extract_features.py index 13962cd..29ec831 100644 --- a/src/extract_features.py +++ b/src/extract_features.py @@ -244,10 +244,8 @@ def create_arguments() -> argparse.Namespace: parser.add_argument('output_path', type=Path, help='Path to the desired output folder') - # Resegmentation path in FAST5 - parser.add_argument('--reseg_path', type=str, default=DEFAULT_RESEGMENTATION_PATH, - help='''Path to resegmentation group in FAST5 file - (default: Analyses/RawGenomeCorrected_000/BaseCalled_template)''') + parser.add_argument('--reference', type=str, required=True, + help='Path to the reference file') parser.add_argument('--norm_method', type=str, default='standardization', help='Function name to use for signal normalization (default: standardization)') @@ -256,9 +254,6 @@ def create_arguments() -> argparse.Namespace: help='''Motif to be searched for in the sequences. Regular expressions can be used. (default: CG)''') - parser.add_argument('--sample_size', type=int, default=20, - help='Sample size for every base in the given k-mer. (default: 20)') - parser.add_argument('--window', type=int, default=8, help='''Window size around central position. Total k-mer size is: K = 2*W + 1. (default: 8)''') From d92ada57fd895c324ffcc8c68b3508ca82b09e9d Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Tue, 1 Jun 2021 03:34:07 +0200 Subject: [PATCH 10/21] Solve the overlapping regions problem --- remove-tombo/basecall.py | 22 ++++ remove-tombo/custom_processor.py | 180 ++++++++++++++----------------- remove-tombo/util.py | 11 ++ 3 files changed, 114 insertions(+), 99 deletions(-) create mode 100644 remove-tombo/basecall.py create mode 100644 remove-tombo/util.py diff --git a/remove-tombo/basecall.py b/remove-tombo/basecall.py new file mode 100644 index 0000000..be936ba --- /dev/null +++ b/remove-tombo/basecall.py @@ -0,0 +1,22 @@ +from pyguppyclient import GuppyBasecallerClient, yield_reads +import numpy as np + + +# config='dna_r9.4.1_450bps_hac' for more precise basecalling +def basecall(files, config='dna_r9.4.1_450bps_fast'): + with GuppyBasecallerClient(config_name=config, trace=True) as client: + for file in files: + for read in yield_reads(file): + called = client.basecall(read)[1] + yield read, called + + +def sequence_to_raw(read, called): + first_signal_id = len(read.signal) - called.trimmed_samples + move_index = np.nonzero(called.move)[0] + + seq_to_raw_start = first_signal_id + move_index * called.model_stride + seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) + seq_to_raw_end = seq_to_raw_start + seq_to_raw_len + + return list(zip(seq_to_raw_start, seq_to_raw_end)) diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index 1f14c8c..e7eaff3 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -1,16 +1,17 @@ from pathlib import Path -from tqdm import tqdm -from pyguppyclient import GuppyBasecallerClient, get_fast5_files, yield_reads -import mappy as mp +from pyguppyclient import get_fast5_files +import mappy from Bio import SeqIO import re +from tqdm import tqdm import numpy as np +from basecall import basecall, sequence_to_raw from util import ResegmentationData def make_aligner(reference_file): - aligner = mp.Aligner(reference_file, preset='map-ont') # Load or build index + aligner = mappy.Aligner(reference_file, preset='map-ont') # Load or build index if not aligner: raise Exception("ERROR: failed to load/build index") @@ -32,27 +33,18 @@ def get_motif_positions(reference, motif, index): fwd_pos = set(m.start() + index for m in fwd_matches) # Reverse strand - rev_matches = re.finditer(motif, mp.revcomp(reference), re.I) + rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) rev_pos = set(r_len - (m.start() + index) - 1 for m in rev_matches) return fwd_pos, rev_pos -# config='dna_r9.4.1_450bps_hac' for more precise basecalling -def basecall(read_file, config='dna_r9.4.1_450bps_fast'): - with GuppyBasecallerClient(config_name=config, trace=True) as client: - for read in yield_reads(read_file): - called = client.basecall(read)[1] - return read, called - - def align(aligner, query, mapq): for hit in aligner.map(query): # Traverse alignments if hit.is_primary: # Check if the alignment is primary if hit.mapq < mapq: # Check if the mapping quality is below set threshold return None return hit - return None @@ -62,130 +54,121 @@ def get_relevant_motif_positions(motif_positions, alignment): relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) if alignment.strand == 1: - return {p - alignment.r_st for p in relevant_positions} + return {pos - alignment.r_st for pos in relevant_positions} else: - return {alignment.r_en - 1 - p for p in relevant_positions} + return {alignment.r_en - 1 - pos for pos in relevant_positions} -def reference_to_basecall(alignment): +def resolve_insertions(alignment, seq_to_raw): cigar = alignment.cigar if alignment.strand == 1 else reversed(alignment.cigar) - ref_to_bc = [] - qpos = alignment.q_st + r_pos, q_pos = 0, alignment.q_st + r_len = alignment.r_en - alignment.r_st + + signal_intervals = [None] * r_len + insertion = False + deletion_idx = [] for length, operation in cigar: if operation in {0, 7, 8}: # Match or mismatch - for i in range(length): - ref_to_bc.append((qpos + i, qpos + i + 1)) - qpos += length - - elif operation == 1: # Insertion - qpos += length + if insertion: + signal_intervals[r_pos] = center, seq_to_raw[q_pos][1] # Base to the right + insertion = False + length -= 1 + r_pos += 1 + q_pos += 1 - elif operation in {2, 3}: # Deletion or skip - for _ in range(length): - ref_to_bc.append((qpos, qpos)) - - return ref_to_bc - - -def sequence_to_raw(read, called): - first_signal_id = len(read.signal) - called.trimmed_samples - move_index = np.nonzero(called.move)[0] - - seq_to_raw_start = first_signal_id + move_index * called.model_stride - seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) - seq_to_raw_end = seq_to_raw_start + seq_to_raw_len + for i in range(length): + signal_intervals[r_pos + i] = seq_to_raw[q_pos + i] - return list(zip(seq_to_raw_start, seq_to_raw_end)) + r_pos += length + q_pos += length + elif operation == 1: # Insertion + insertion_interval = seq_to_raw[q_pos][0], seq_to_raw[q_pos + length][0] -def resolve_insertions(position, ref_to_bc, seq_to_raw, window): - base_intervals = ref_to_bc[position - window: position + window + 1] # End is exclusive - signal_intervals = seq_to_raw[base_intervals[0][0]: base_intervals[-1][1]] + center = int(np.mean(insertion_interval)) + signal_intervals[r_pos - 1] = signal_intervals[r_pos - 1][0], center # Base to the left + insertion = True - deletion_idx = [] + q_pos += length - for i in range(len(base_intervals)): - if base_intervals[i][0] == base_intervals[i][1]: # Deletion or skip - signal_intervals.insert(i, (seq_to_raw[base_intervals[i][0]][0], seq_to_raw[base_intervals[i][0]][0])) + elif operation in {2, 3}: # Deletion or skip + deletion_idx.append((r_pos, r_pos + length)) - if deletion_idx and i == deletion_idx[-1][1]: - deletion_idx[-1] = deletion_idx[-1][0], i + 1 - else: - deletion_idx.append((i, i + 1)) + if insertion: + signal_intervals[r_pos] = center, seq_to_raw[q_pos][0] # Base to the right + insertion = False + length -= 1 + r_pos += 1 - if i != len(base_intervals) - 1 and base_intervals[i][1] != base_intervals[i + 1][0]: # Resolve insertions - no_of_insertions = base_intervals[i + 1][0] - base_intervals[i][1] - insertion_interval = signal_intervals[i][1], signal_intervals[i + 1 + no_of_insertions][0] + for i in range(length): + signal_intervals[r_pos + i] = seq_to_raw[q_pos][0], seq_to_raw[q_pos][0] - center = int(np.mean(insertion_interval)) - del signal_intervals[i + 1: i + 1 + no_of_insertions] + r_pos += length - signal_intervals[i] = signal_intervals[i][0], center # Base to the left - signal_intervals[i + 1] = center, signal_intervals[i + 1][1] # Base to the right + else: + raise ValueError('Invalid CIGAR operation') - return base_intervals, signal_intervals, deletion_idx + return signal_intervals, deletion_idx def resolve_deletions(signal_intervals, deletion_idx): - for idx_st, idx_en in deletion_idx: # Resolve deletions - if idx_st == 0 and idx_en == len(signal_intervals): # Region consisting only of deletions - break - - if idx_st == 0: - start, end = idx_st, idx_en - elif idx_en == len(signal_intervals): - start, end = idx_st - 1, idx_en - 1 - else: - start, end = idx_st - 1, idx_en - - sig_st, sig_en = signal_intervals[start][0], signal_intervals[end][1] - intervals = np.array_split(range(sig_st, sig_en), end - start + 1) + for idx_st, idx_en in deletion_idx: + sig_st, sig_en = signal_intervals[idx_st - 1][0], signal_intervals[idx_en][1] + intervals = np.array_split(range(sig_st, sig_en), idx_en - idx_st + 2) + + if len(intervals[-1]) == 0: # If there is not enough signal points to divide among bases + while len(intervals[-1]) == 0: + intervals.pop(-1) + interval = intervals.pop(-1) + signal_intervals[idx_en] = interval[0], interval[-1] + 1 # Base to the right must have > 0 signal points + + for i in range(idx_st - 1, idx_en + 1): + if len(intervals) == 0: # Bases in the middle (deletions) which have 0 signal points + if i < idx_en: + signal_intervals[i] = signal_intervals[idx_en][0], signal_intervals[idx_en][0] + continue - for i in range(start, end + 1): interval = intervals.pop(0) - if len(interval) == 0: # If there is not enough signal points for the remaining bases - break signal_intervals[i] = interval[0], interval[-1] + 1 return signal_intervals -def resolve_region(position, ref_to_bc, seq_to_raw, window): - if position - 8 < 0 or position + 8 >= len(ref_to_bc): - return None - - base_intervals, signal_intervals, deletion_idx = resolve_insertions(position, ref_to_bc, seq_to_raw, window) - signal_intervals = resolve_deletions(signal_intervals, deletion_idx) - - assert len(base_intervals) == len(signal_intervals) - - return signal_intervals - - -def custom_processor(fast5_file, aligner, reference, motif_positions, mapq, window): - read, called = basecall(fast5_file) +def custom_processor(basecall_data, aligner, reference, motif_positions, mapq, window): + read, called = basecall_data alignment = align(aligner, called.seq, mapq) if not alignment: return None relevant_motif_positions = get_relevant_motif_positions(motif_positions, alignment) + if not relevant_motif_positions: + return None - ref_to_bc = reference_to_basecall(alignment) seq_to_raw = sequence_to_raw(read, called) + signal_intervals, deletion_idx = resolve_insertions(alignment, seq_to_raw) + signal_intervals = resolve_deletions(signal_intervals, deletion_idx) + + resegmentation_data = [] + for motif_position in relevant_motif_positions: - event_intervals = resolve_region(motif_position, ref_to_bc, seq_to_raw, window) - if event_intervals is None: + r_len = alignment.r_en - alignment.r_st + if motif_position - window < 0 or motif_position + window >= r_len: continue - position = alignment.r_st + motif_position + position = alignment.r_st + motif_position if alignment.strand == 1 else alignment.r_en - 1 - motif_position + event_intervals = signal_intervals[motif_position - window: motif_position + window + 1] event_lens = [end - start for start, end in event_intervals] bases = reference[position - window: position + window + 1] - return ResegmentationData(position, event_intervals, event_lens, bases) + assert len(event_intervals) == len(event_lens) == len(bases) + + resegmentation_data.append(ResegmentationData(position, event_intervals, event_lens, bases)) + + return resegmentation_data def process_data(dir_in, reference_file, mapq=0, motif='CG', index=0, window=8): @@ -195,12 +178,11 @@ def process_data(dir_in, reference_file, mapq=0, motif='CG', index=0, window=8): reference = get_reference(reference_file) motif_positions = get_motif_positions(reference, motif, index) - for fast5_file in tqdm(fast5_files): - resegmentation_data = custom_processor(fast5_file, aligner, reference, motif_positions, mapq, window) - if resegmentation_data is None: - continue + for basecall_data in tqdm(basecall(fast5_files)): + resegmentation_data = custom_processor(basecall_data, aligner, reference, motif_positions, mapq, window) - print(resegmentation_data) + if resegmentation_data: + print(resegmentation_data) if __name__ == '__main__': diff --git a/remove-tombo/util.py b/remove-tombo/util.py new file mode 100644 index 0000000..93f3369 --- /dev/null +++ b/remove-tombo/util.py @@ -0,0 +1,11 @@ +from dataclasses import dataclass +from typing import List, Tuple +import numpy as np + + +@dataclass +class ResegmentationData: + position: int + event_intervals: List[Tuple] # Intervals of signal points + event_lens: np.ndarray # Lengths of intervals of signal points + bases: str From dc3a0a0949b595fd10d2ec221fa447b909e7c0c3 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Tue, 1 Jun 2021 16:48:14 +0200 Subject: [PATCH 11/21] Added plots for signal points ratios --- remove-tombo/plots/ratio_D_mod.png | Bin 0 -> 19908 bytes remove-tombo/plots/ratio_D_nomod.png | Bin 0 -> 20257 bytes 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Sanja Deur Date: Tue, 1 Jun 2021 20:45:15 +0200 Subject: [PATCH 12/21] Code enhancement --- remove-tombo/basecall.py | 10 +++++-- remove-tombo/custom_processor.py | 47 +++++++++++++++++--------------- remove-tombo/util.py | 9 ++++-- 3 files changed, 39 insertions(+), 27 deletions(-) diff --git a/remove-tombo/basecall.py b/remove-tombo/basecall.py index be936ba..ce0f95a 100644 --- a/remove-tombo/basecall.py +++ b/remove-tombo/basecall.py @@ -1,9 +1,13 @@ from pyguppyclient import GuppyBasecallerClient, yield_reads import numpy as np +from typing import List + +from util import Interval + # config='dna_r9.4.1_450bps_hac' for more precise basecalling -def basecall(files, config='dna_r9.4.1_450bps_fast'): +def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): with GuppyBasecallerClient(config_name=config, trace=True) as client: for file in files: for read in yield_reads(file): @@ -11,7 +15,7 @@ def basecall(files, config='dna_r9.4.1_450bps_fast'): yield read, called -def sequence_to_raw(read, called): +def sequence_to_raw(read, called) -> List[Interval]: first_signal_id = len(read.signal) - called.trimmed_samples move_index = np.nonzero(called.move)[0] @@ -19,4 +23,4 @@ def sequence_to_raw(read, called): seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) seq_to_raw_end = seq_to_raw_start + seq_to_raw_len - return list(zip(seq_to_raw_start, seq_to_raw_end)) + return [Interval(st, en) for st, en in zip(seq_to_raw_start, seq_to_raw_end)] diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index e7eaff3..fd18fe8 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -6,11 +6,13 @@ from tqdm import tqdm import numpy as np +from typing import Tuple, Set, List, Optional + from basecall import basecall, sequence_to_raw -from util import ResegmentationData +from util import Interval, ResegmentationData -def make_aligner(reference_file): +def make_aligner(reference_file: str) -> mappy.Aligner: aligner = mappy.Aligner(reference_file, preset='map-ont') # Load or build index if not aligner: raise Exception("ERROR: failed to load/build index") @@ -18,14 +20,14 @@ def make_aligner(reference_file): return aligner -def get_reference(reference_file): +def get_reference(reference_file: str) -> str: for seq_record in SeqIO.parse(reference_file, 'fasta'): reference = str(seq_record.seq) return reference -def get_motif_positions(reference, motif, index): +def get_motif_positions(reference: str, motif: str, index: int) -> Tuple[Set[int], Set[int]]: r_len = len(reference) # Forward strand @@ -39,7 +41,7 @@ def get_motif_positions(reference, motif, index): return fwd_pos, rev_pos -def align(aligner, query, mapq): +def align(aligner: mappy.Aligner, query: str, mapq: int) -> Optional[mappy.Alignment]: for hit in aligner.map(query): # Traverse alignments if hit.is_primary: # Check if the alignment is primary if hit.mapq < mapq: # Check if the mapping quality is below set threshold @@ -48,7 +50,7 @@ def align(aligner, query, mapq): return None -def get_relevant_motif_positions(motif_positions, alignment): +def get_relevant_motif_positions(motif_positions: Tuple[Set[int], Set[int]], alignment: mappy.Alignment) -> Set[int]: strand_pos = motif_positions[0] if alignment.strand == 1 else motif_positions[1] relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) @@ -59,7 +61,7 @@ def get_relevant_motif_positions(motif_positions, alignment): return {alignment.r_en - 1 - pos for pos in relevant_positions} -def resolve_insertions(alignment, seq_to_raw): +def resolve_insertions(alignment: mappy.Alignment, seq_to_raw: List[Interval]) -> Tuple[List[Interval], List[Interval]]: cigar = alignment.cigar if alignment.strand == 1 else reversed(alignment.cigar) r_pos, q_pos = 0, alignment.q_st @@ -72,7 +74,7 @@ def resolve_insertions(alignment, seq_to_raw): for length, operation in cigar: if operation in {0, 7, 8}: # Match or mismatch if insertion: - signal_intervals[r_pos] = center, seq_to_raw[q_pos][1] # Base to the right + signal_intervals[r_pos] = Interval(center, seq_to_raw[q_pos].end) # Base to the right insertion = False length -= 1 r_pos += 1 @@ -85,25 +87,25 @@ def resolve_insertions(alignment, seq_to_raw): q_pos += length elif operation == 1: # Insertion - insertion_interval = seq_to_raw[q_pos][0], seq_to_raw[q_pos + length][0] - + insertion_interval = Interval(seq_to_raw[q_pos].start, seq_to_raw[q_pos + length].start) center = int(np.mean(insertion_interval)) - signal_intervals[r_pos - 1] = signal_intervals[r_pos - 1][0], center # Base to the left + + signal_intervals[r_pos - 1] = Interval(signal_intervals[r_pos - 1].start, center) # Base to the left insertion = True q_pos += length elif operation in {2, 3}: # Deletion or skip - deletion_idx.append((r_pos, r_pos + length)) + deletion_idx.append(Interval(r_pos, r_pos + length)) if insertion: - signal_intervals[r_pos] = center, seq_to_raw[q_pos][0] # Base to the right + signal_intervals[r_pos] = Interval(center, seq_to_raw[q_pos].start) # Base to the right insertion = False length -= 1 r_pos += 1 for i in range(length): - signal_intervals[r_pos + i] = seq_to_raw[q_pos][0], seq_to_raw[q_pos][0] + signal_intervals[r_pos + i] = Interval(seq_to_raw[q_pos].start, seq_to_raw[q_pos].start) r_pos += length @@ -113,30 +115,31 @@ def resolve_insertions(alignment, seq_to_raw): return signal_intervals, deletion_idx -def resolve_deletions(signal_intervals, deletion_idx): +def resolve_deletions(signal_intervals: List[Interval], deletion_idx: List[Interval]) -> List[Interval]: for idx_st, idx_en in deletion_idx: - sig_st, sig_en = signal_intervals[idx_st - 1][0], signal_intervals[idx_en][1] + sig_st, sig_en = signal_intervals[idx_st - 1].start, signal_intervals[idx_en].end intervals = np.array_split(range(sig_st, sig_en), idx_en - idx_st + 2) if len(intervals[-1]) == 0: # If there is not enough signal points to divide among bases while len(intervals[-1]) == 0: intervals.pop(-1) interval = intervals.pop(-1) - signal_intervals[idx_en] = interval[0], interval[-1] + 1 # Base to the right must have > 0 signal points + signal_intervals[idx_en] = Interval(interval[0], interval[-1] + 1) # Base to the right must have > 0 signal points for i in range(idx_st - 1, idx_en + 1): if len(intervals) == 0: # Bases in the middle (deletions) which have 0 signal points if i < idx_en: - signal_intervals[i] = signal_intervals[idx_en][0], signal_intervals[idx_en][0] + signal_intervals[i] = Interval(signal_intervals[idx_en].start, signal_intervals[idx_en].start) continue interval = intervals.pop(0) - signal_intervals[i] = interval[0], interval[-1] + 1 + signal_intervals[i] = Interval(interval[0], interval[-1] + 1) return signal_intervals -def custom_processor(basecall_data, aligner, reference, motif_positions, mapq, window): +def custom_processor(basecall_data: Tuple, aligner: mappy.Aligner, reference: str, motif_positions: Set[int], + mapq: int, window: int) -> ResegmentationData: read, called = basecall_data alignment = align(aligner, called.seq, mapq) @@ -161,7 +164,7 @@ def custom_processor(basecall_data, aligner, reference, motif_positions, mapq, w position = alignment.r_st + motif_position if alignment.strand == 1 else alignment.r_en - 1 - motif_position event_intervals = signal_intervals[motif_position - window: motif_position + window + 1] - event_lens = [end - start for start, end in event_intervals] + event_lens = [interval.end - interval.start for interval in event_intervals] bases = reference[position - window: position + window + 1] assert len(event_intervals) == len(event_lens) == len(bases) @@ -171,7 +174,7 @@ def custom_processor(basecall_data, aligner, reference, motif_positions, mapq, w return resegmentation_data -def process_data(dir_in, reference_file, mapq=0, motif='CG', index=0, window=8): +def process_data(dir_in: Path, reference_file: str, mapq: int = 0, motif: str = 'CG', index: int = 0, window: int = 8): fast5_files = get_fast5_files(dir_in, recursive=True) aligner = make_aligner(reference_file) diff --git a/remove-tombo/util.py b/remove-tombo/util.py index 93f3369..637823b 100644 --- a/remove-tombo/util.py +++ b/remove-tombo/util.py @@ -1,11 +1,16 @@ +from collections import namedtuple from dataclasses import dataclass -from typing import List, Tuple import numpy as np +from typing import List + + +Interval = namedtuple('Interval', ['start', 'end']) # Start - inclusive, end - exclusive + @dataclass class ResegmentationData: position: int - event_intervals: List[Tuple] # Intervals of signal points + event_intervals: List[Interval] # Intervals of signal points event_lens: np.ndarray # Lengths of intervals of signal points bases: str From 56879174f178832d4733ec538e28531ca1e39e55 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Tue, 1 Jun 2021 21:01:12 +0200 Subject: [PATCH 13/21] Added argument parser --- remove-tombo/custom_processor.py | 27 +++++++++++++++++++++------ 1 file changed, 21 insertions(+), 6 deletions(-) diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index fd18fe8..6dea686 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -1,3 +1,4 @@ +import argparse from pathlib import Path from pyguppyclient import get_fast5_files import mappy @@ -174,8 +175,9 @@ def custom_processor(basecall_data: Tuple, aligner: mappy.Aligner, reference: st return resegmentation_data -def process_data(dir_in: Path, reference_file: str, mapq: int = 0, motif: str = 'CG', index: int = 0, window: int = 8): - fast5_files = get_fast5_files(dir_in, recursive=True) +def process_data(input_path: str, recursive: bool, reference_file: str, + mapq: int = 0, motif: str = 'CG', index: int = 0, window: int = 8): + fast5_files = get_fast5_files(input_path, recursive=recursive) aligner = make_aligner(reference_file) reference = get_reference(reference_file) @@ -188,9 +190,22 @@ def process_data(dir_in: Path, reference_file: str, mapq: int = 0, motif: str = print(resegmentation_data) +def parse_arguments(): + parser = argparse.ArgumentParser(description='Custom processor') + + parser.add_argument('-i', '--input_path', type=str, required=True, + help='Path to the input file or folder containing FAST5 files') + + parser.add_argument('-r', '--recursive', action='store_true', + help='Flag to indicate if folder will be searched recursively (default: False)') + + parser.add_argument('--reference', type=str, required=True, + help='Path to the reference file') + + return parser.parse_args() + + if __name__ == '__main__': - modification = 'mod' # 'mod' or 'nomod' - dir_in = Path(f'/home/sdeur/data/{modification}') - reference_file = '/home/sdeur/data/ecoli_k12_mg1655.fasta' + args = parse_arguments() - process_data(dir_in, reference_file) + process_data(args.input_path, args.recursive, args.reference) From 4af60361548997e94d898cf0b8747556d6bf1d51 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 2 Jun 2021 03:05:21 +0200 Subject: [PATCH 14/21] Custom processor written as a class --- remove-tombo/custom_processor.py | 8 +- src/processor/alignment.py | 34 +++++++ src/processor/basecall.py | 26 +++++ src/processor/custom_processor.py | 156 ++++++++++++++++++++++++++++++ src/processor/util.py | 16 +++ 5 files changed, 237 insertions(+), 3 deletions(-) create mode 100644 src/processor/alignment.py create mode 100644 src/processor/basecall.py create mode 100644 src/processor/custom_processor.py create mode 100644 src/processor/util.py diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index 6dea686..5013a93 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -1,5 +1,4 @@ import argparse -from pathlib import Path from pyguppyclient import get_fast5_files import mappy from Bio import SeqIO @@ -164,9 +163,12 @@ def custom_processor(basecall_data: Tuple, aligner: mappy.Aligner, reference: st continue position = alignment.r_st + motif_position if alignment.strand == 1 else alignment.r_en - 1 - motif_position + event_intervals = signal_intervals[motif_position - window: motif_position + window + 1] - event_lens = [interval.end - interval.start for interval in event_intervals] - bases = reference[position - window: position + window + 1] + event_lens = np.array([interval.end - interval.start for interval in event_intervals]) + + region = reference[position - window: position + window + 1] + bases = region if alignment.strand == 1 else mappy.revcomp(region) assert len(event_intervals) == len(event_lens) == len(bases) diff --git a/src/processor/alignment.py b/src/processor/alignment.py new file mode 100644 index 0000000..230d065 --- /dev/null +++ b/src/processor/alignment.py @@ -0,0 +1,34 @@ +import mappy +from Bio import SeqIO +import re + +from typing import Tuple, Set + + +def make_aligner(reference_file: str) -> mappy.Aligner: + aligner = mappy.Aligner(reference_file, preset='map-ont') # Load or build index + if not aligner: + raise Exception("ERROR: failed to load/build index") + + return aligner + + +def get_reference(reference_file: str) -> str: + for seq_record in SeqIO.parse(reference_file, 'fasta'): + reference = str(seq_record.seq) + + return reference + + +def get_motif_positions(reference: str, motif: str, index: int) -> Tuple[Set[int], Set[int]]: + r_len = len(reference) + + # Forward strand + fwd_matches = re.finditer(motif, reference, re.I) + fwd_pos = set(m.start() + index for m in fwd_matches) + + # Reverse strand + rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) + rev_pos = set(r_len - (m.start() + index) - 1 for m in rev_matches) + + return fwd_pos, rev_pos \ No newline at end of file diff --git a/src/processor/basecall.py b/src/processor/basecall.py new file mode 100644 index 0000000..28ce628 --- /dev/null +++ b/src/processor/basecall.py @@ -0,0 +1,26 @@ +from pyguppyclient import GuppyBasecallerClient, yield_reads +import numpy as np + +from typing import List + +from .util import Interval + + +# config='dna_r9.4.1_450bps_hac' for more precise basecalling +def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): + with GuppyBasecallerClient(config_name=config, trace=True) as client: + for file in files: + for read in yield_reads(file): + called = client.basecall(read)[1] + yield read, called + + +def sequence_to_raw(read, called) -> List[Interval]: + first_signal_id = len(read.signal) - called.trimmed_samples + move_index = np.nonzero(called.move)[0] + + seq_to_raw_start = first_signal_id + move_index * called.model_stride + seq_to_raw_len = np.diff(seq_to_raw_start, append=len(read.signal)) + seq_to_raw_end = seq_to_raw_start + seq_to_raw_len + + return [Interval(st, en) for st, en in zip(seq_to_raw_start, seq_to_raw_end)] diff --git a/src/processor/custom_processor.py b/src/processor/custom_processor.py new file mode 100644 index 0000000..8b9a3f0 --- /dev/null +++ b/src/processor/custom_processor.py @@ -0,0 +1,156 @@ +import mappy + +from typing import Tuple, Set, Optional + +from .alignment import make_aligner, get_reference, get_motif_positions +from .basecall import sequence_to_raw +from .util import * + + +class CustomProcessor: + def __init__(self, + basecall_data, + reference_file: str, + mapq: int = 0, + motif: str = 'CG', + index: int = 0, + window: int = 8): + self.basecall_data = basecall_data + self.mapq = mapq + self.window = window + + self.aligner = make_aligner(reference_file) + self.reference = get_reference(reference_file) + self.motif_positions = get_motif_positions(self.reference, motif, index) + + def align(self, query: str) -> Optional[mappy.Alignment]: + for hit in self.aligner.map(query): # Traverse alignments + if hit.is_primary: # Check if the alignment is primary + if hit.mapq < self.mapq: # Check if the mapping quality is below set threshold + return None + return hit + return None + + def _get_relevant_motif_positions(self, alignment: mappy.Alignment) -> Set[int]: + strand_pos = self.motif_positions[0] if alignment.strand == 1 else self.motif_positions[1] + + relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) + + if alignment.strand == 1: + return {pos - alignment.r_st for pos in relevant_positions} + else: + return {alignment.r_en - 1 - pos for pos in relevant_positions} + + @staticmethod + def resolve_insertions(alignment: mappy.Alignment, seq_to_raw: List[Interval]) -> Tuple[List[Interval], List[Interval]]: + cigar = alignment.cigar if alignment.strand == 1 else reversed(alignment.cigar) + + r_pos, q_pos = 0, alignment.q_st + r_len = alignment.r_en - alignment.r_st + + signal_intervals = [None] * r_len + insertion = False + deletion_idx = [] + + for length, operation in cigar: + if operation in {0, 7, 8}: # Match or mismatch + if insertion: + signal_intervals[r_pos] = Interval(center, seq_to_raw[q_pos].end) # Base to the right + insertion = False + length -= 1 + r_pos += 1 + q_pos += 1 + + for i in range(length): + signal_intervals[r_pos + i] = seq_to_raw[q_pos + i] + + r_pos += length + q_pos += length + + elif operation == 1: # Insertion + insertion_interval = Interval(seq_to_raw[q_pos].start, seq_to_raw[q_pos + length].start) + center = int(np.mean(insertion_interval)) + + signal_intervals[r_pos - 1] = Interval(signal_intervals[r_pos - 1].start, center) # Base to the left + insertion = True + + q_pos += length + + elif operation in {2, 3}: # Deletion or skip + deletion_idx.append(Interval(r_pos, r_pos + length)) + + if insertion: + signal_intervals[r_pos] = Interval(center, seq_to_raw[q_pos].start) # Base to the right + insertion = False + length -= 1 + r_pos += 1 + + for i in range(length): + signal_intervals[r_pos + i] = Interval(seq_to_raw[q_pos].start, seq_to_raw[q_pos].start) + + r_pos += length + + else: + raise ValueError('Invalid CIGAR operation') + + return signal_intervals, deletion_idx + + @staticmethod + def resolve_deletions(signal_intervals: List[Interval], deletion_idx: List[Interval]) -> List[Interval]: + for idx_st, idx_en in deletion_idx: + sig_st, sig_en = signal_intervals[idx_st - 1].start, signal_intervals[idx_en].end + intervals = np.array_split(range(sig_st, sig_en), idx_en - idx_st + 2) + + if len(intervals[-1]) == 0: # If there is not enough signal points to divide among bases + while len(intervals[-1]) == 0: + intervals.pop(-1) + interval = intervals.pop(-1) + signal_intervals[idx_en] = Interval(interval[0], interval[-1] + 1) # Base to the right must have > 0 signal points + + for i in range(idx_st - 1, idx_en + 1): + if len(intervals) == 0: # Bases in the middle (deletions) which have 0 signal points + if i < idx_en: + signal_intervals[i] = Interval(signal_intervals[idx_en].start, signal_intervals[idx_en].start) + continue + + interval = intervals.pop(0) + signal_intervals[i] = Interval(interval[0], interval[-1] + 1) + + return signal_intervals + + def process(self) -> Optional[List[ResegmentationData]]: + read, called = self.basecall_data + + alignment = self.align(called.seq) + if not alignment: + return None + + relevant_motif_positions = self._get_relevant_motif_positions(alignment) + if not relevant_motif_positions: + return None + + seq_to_raw = sequence_to_raw(read, called) + + signal_intervals, deletion_idx = CustomProcessor.resolve_insertions(alignment, seq_to_raw) + signal_intervals = CustomProcessor.resolve_deletions(signal_intervals, deletion_idx) + + resegmentation_data = [] + + for motif_position in relevant_motif_positions: + r_len = alignment.r_en - alignment.r_st + if motif_position - self.window < 0 or motif_position + self.window >= r_len: + continue + + position = alignment.r_st + motif_position if alignment.strand == 1 else alignment.r_en - 1 - motif_position + + event_intervals = signal_intervals[motif_position - self.window: motif_position + self.window + 1] + event_lens = np.array([interval.end - interval.start for interval in event_intervals]) + + region = self.reference[position - self.window: position + self.window + 1] + bases = region if alignment.strand == 1 else mappy.revcomp(region) + + assert len(event_intervals) == len(event_lens) == len(bases) + + resegmentation_data.append(ResegmentationData(position, event_intervals, event_lens, bases)) + + return resegmentation_data diff --git a/src/processor/util.py b/src/processor/util.py new file mode 100644 index 0000000..637823b --- /dev/null +++ b/src/processor/util.py @@ -0,0 +1,16 @@ +from collections import namedtuple +from dataclasses import dataclass +import numpy as np + +from typing import List + + +Interval = namedtuple('Interval', ['start', 'end']) # Start - inclusive, end - exclusive + + +@dataclass +class ResegmentationData: + position: int + event_intervals: List[Interval] # Intervals of signal points + event_lens: np.ndarray # Lengths of intervals of signal points + bases: str From 7265f5dedd54bacb5adeafb6b6634478e1ab96ee Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 2 Jun 2021 03:22:28 +0200 Subject: [PATCH 15/21] Rewrite extract features --- remove-tombo/basecall.py | 7 +- remove-tombo/custom_processor.py | 5 +- src/extract_features.py | 125 ++++++++++++++++++++---------- src/processor/basecall.py | 7 +- src/processor/custom_processor.py | 8 +- 5 files changed, 99 insertions(+), 53 deletions(-) diff --git a/remove-tombo/basecall.py b/remove-tombo/basecall.py index ce0f95a..189e151 100644 --- a/remove-tombo/basecall.py +++ b/remove-tombo/basecall.py @@ -1,13 +1,14 @@ from pyguppyclient import GuppyBasecallerClient, yield_reads +from pyguppyclient.decode import ReadData, CalledReadData import numpy as np -from typing import List +from typing import Tuple, List from util import Interval # config='dna_r9.4.1_450bps_hac' for more precise basecalling -def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): +def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast') -> Tuple[ReadData, CalledReadData]: with GuppyBasecallerClient(config_name=config, trace=True) as client: for file in files: for read in yield_reads(file): @@ -15,7 +16,7 @@ def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): yield read, called -def sequence_to_raw(read, called) -> List[Interval]: +def sequence_to_raw(read: ReadData, called: CalledReadData) -> List[Interval]: first_signal_id = len(read.signal) - called.trimmed_samples move_index = np.nonzero(called.move)[0] diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index 5013a93..8f98e24 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -1,5 +1,6 @@ import argparse from pyguppyclient import get_fast5_files +from pyguppyclient.decode import ReadData, CalledReadData import mappy from Bio import SeqIO import re @@ -138,8 +139,8 @@ def resolve_deletions(signal_intervals: List[Interval], deletion_idx: List[Inter return signal_intervals -def custom_processor(basecall_data: Tuple, aligner: mappy.Aligner, reference: str, motif_positions: Set[int], - mapq: int, window: int) -> ResegmentationData: +def custom_processor(basecall_data: Tuple[ReadData, CalledReadData], aligner: mappy.Aligner, reference: str, + motif_positions: Set[int], mapq: int, window: int) -> ResegmentationData: read, called = basecall_data alignment = align(aligner, called.seq, mapq) diff --git a/src/extract_features.py b/src/extract_features.py index 29ec831..0abe351 100644 --- a/src/extract_features.py +++ b/src/extract_features.py @@ -2,10 +2,12 @@ import sys import time +import numpy as np from tqdm import tqdm from pathlib import Path import traceback from concurrent.futures import as_completed +from pyguppyclient.decode import ReadData, CalledReadData import argparse @@ -14,6 +16,10 @@ from utils.parallel_processing import get_executor from utils.writer import BinaryWriter +from processor.basecall import basecall +from processor.custom_processor import CustomProcessor +from processor.util import Interval + BATCH_SIZE = 4_000 @@ -41,8 +47,9 @@ def get_info_args(args: argparse.Namespace) -> Dict[str, Any]: """ info_args = { 'norm_method': args.norm_method, + 'mapq': args.mapq, 'motif': args.motif, - 'sample_size': args.sample_size, + 'index': args.index, 'window': args.window, 'label': args.label } @@ -50,17 +57,40 @@ def get_info_args(args: argparse.Namespace) -> Dict[str, Any]: return info_args -def generate_data(read: Read, +def get_raw_signal(read: ReadData, event_interval: Interval, norm_method, continuous: bool=True) -> np.ndarray: + """ Returns the raw signal for the read. + + Returns the raw signal for the given read. Optionally, it converts discrete signal to continuous signal. + + :param read: Basecalled read + :param event_interval: Interval of signal points + :param norm_method: Signal normalization method + :param continuous: True if returned signal should be continuous, otherwise False + :return: Raw signal for the given read + """ + signal = read.signal[event_interval.start: event_interval.end] + + if continuous: + signal = read.daq_scaling * (signal + read.daq_offset) + + norm_func = normalization_factory(norm_method) + if norm_func: + signal = norm_func(signal) + + return signal + + +def generate_data(read: ReadData, reseg_data: List[ResegmentationData], - sample_size: int=20) -> Optional[List[Example]]: + norm_method: str) -> Optional[List[Example]]: """ Function that generates data for the specified read. This function generates list of examples for the given read. Data is generated from raw signal for the given read, - and information extracted from resegmentation group. + and information extracted from resegmentation data. :param read: Read for which examples will be generated - :param reseg_data: Resegmentation data extracted from the resegmentation group - :param sample_size: Number of sample points per base + :param reseg_data: Resegmentation data + :param norm_method: Signal normalization method :return: List of generated examples for the given read and resegmentation data """ all_examples = [] @@ -69,7 +99,7 @@ def generate_data(read: Read, example_points = [] for interval in reseg_example.event_intervals: - points, _ = read.signal_for_interval(interval, sample_size) + points = get_raw_signal(read, interval, norm_method) example_points.append(points) example_points = np.concatenate(example_points) @@ -84,39 +114,43 @@ def generate_data(read: Read, def process_read( - path: Path, - reseg_path: Optional[str]=None, + basecall_data: Tuple[ReadData, CalledReadData], + reference: str, norm_method: Optional[str]='standardization', + mapq: int = 10, motif: str='CG', - sample_size: int=20, + index: int=0, window: int=8, bed_pos: Optional[Set[GenomicPos]]=None) -> Optional[FeaturesData]: """ This function processes the given read to generate data. - This function extracts resegmentation information, samples the signal and extracts alignment data. + This function extracts resegmentation information, and extracts alignment data. - :param path: Path to the single FAST5 read - :param reseg_path: Resegmentation path in re-segmented FAST5 file + :param basecall_data: Basecalled data: (read, called) + :param reference: Path to the reference file :param norm_method: Signal normalization method + :param mapq: Mapping quality threshold :param motif: Motif for which positions will be extracted - :param sample_size: Number of sample points per base + :param index: Index of the central position in the motif :param window: Size of left and right windows around central position :param bed_pos: Positions used for filtering motif positions :return: FeaturesData object if at least one example is present, otherwise None """ - with h5py.File(str(path), 'r', libver='latest') as fd: - read = Read(fd, norm_method) + processor = CustomProcessor(basecall_data, reference, mapq, motif, index, window) + resegmentation_data = processor.process() + if not resegmentation_data: + return - reseg_processor = ResegmentationProcessor(read, reseg_path) - event_pos = reseg_processor.motif_positions(motif, window, bed_pos) - reseg_data = reseg_processor.get_resegmentation_data(event_pos) + read, called = basecall_data - examples = generate_data(read, reseg_data, sample_size) - if not examples: - return + examples = generate_data(read, resegmentation_data, norm_method) + if not examples: + return - align_data = reseg_processor.get_alignment_data() - return FeaturesData(align_data.chromosome, align_data.strand, read.get_read_id(), examples) + align_data = processor.align(called.seq) + strand = Strand.strand_from_str('+' if align_data.strand == 1 else '-') + + return FeaturesData(align_data.ctg, strand, read.read_id, examples) def error_callback(path, exception): @@ -127,27 +161,28 @@ def error_callback(path, exception): def init_workers( info_args: Dict[str, Any], - reseg_path: Optional[str]=None, norm_method: Optional[str]='standardization', + mapq: int=10, motif: str='CG', - sample_size: int=20, + index: int=0, window: int=8, bed_data: Optional[BEDData]=None) -> None: # Initializing workers with constant arguments - global _INFO_ARGS, _RESEG_PATH, _NORM_METHOD, _MOTIF, _SAMPLE_SIZE, _WINDOW, _BED_DATA + global _INFO_ARGS, _NORM_METHOD, _MAPQ, _MOTIF, _INDEX, _WINDOW, _BED_DATA _INFO_ARGS = info_args - _RESEG_PATH = reseg_path _NORM_METHOD = norm_method + _MAPQ = mapq _MOTIF = motif - _SAMPLE_SIZE = sample_size + _INDEX = index _WINDOW = window _BED_DATA = bed_data -def worker_process_reads(paths: List[Path], out_path: Path) -> Tuple[Path, int]: +def worker_process_reads(paths: List[Path], reference: str, out_path: Path) -> Tuple[Path, int]: """ Function that processes input file list and stores generated data :param paths: List of input files that will be processed + :param reference: Path to the reference file :param out_path: Path to the generated output :return: Path and number of failed files """ @@ -156,9 +191,9 @@ def worker_process_reads(paths: List[Path], out_path: Path) -> Tuple[Path, int]: with BinaryWriter(str(out_path)) as writer: bed_pos = set(_BED_DATA.keys()) if _BED_DATA is not None else None - for path in paths: + for path in tqdm(basecall(paths)): try: - result = process_read(path, _RESEG_PATH, _NORM_METHOD, _MOTIF, _SAMPLE_SIZE, _WINDOW, bed_pos) + result = process_read(path, reference, _NORM_METHOD, _MAPQ, _MOTIF, _INDEX, _WINDOW, bed_pos) if result is not None: writer.write_data(result, _BED_DATA, _INFO_ARGS['label']) @@ -204,7 +239,7 @@ def process_data(args: argparse.Namespace) -> None: info_path = Path(args.output_path, 'info.txt') BinaryWriter.write_extraction_info(info_path, info_args) - workers_args = (info_args, args.reseg_path, args.norm_method, args.motif, args.sample_size, args.window, bed_info) + workers_args = (info_args, args.norm_method, args.mapq, args.motif, args.index, args.window, bed_info) with get_executor(args.workers, initializer=init_workers, initargs=workers_args) as executor: futures = [] @@ -218,7 +253,7 @@ def process_data(args: argparse.Namespace) -> None: out_file_path = Path(args.output_path, f'{i+1}.bin.tmp') - future = executor.submit(worker_process_reads, files[start:end], out_file_path) + future = executor.submit(worker_process_reads, files[start:end], args.reference, out_file_path) futures.append(future) tqdm_with_time('Extracting features', last_action_time) @@ -238,25 +273,31 @@ def create_arguments() -> argparse.Namespace: # Input and output arguments parser.add_argument('input_path', type=Path, help='Path to the input file or folder containing FAST5 files') + parser.add_argument('-r', '--recursive', action='store_true', - help='''Flag to indicate if folder will be searched recursively - (default: False)''') + help='Flag to indicate if folder will be searched recursively (default: False)') + parser.add_argument('output_path', type=Path, help='Path to the desired output folder') + # Other arguments parser.add_argument('--reference', type=str, required=True, help='Path to the reference file') parser.add_argument('--norm_method', type=str, default='standardization', help='Function name to use for signal normalization (default: standardization)') + parser.add_argument('--mapq', type=int, default=10, + help='Mapping quality threshold (default: 10)') + parser.add_argument('--motif', type=str, default='CG', - help='''Motif to be searched for in the sequences. - Regular expressions can be used. (default: CG)''') + help='Motif to be searched for in the sequences. Regular expressions can be used (default: CG)') + + parser.add_argument('--index', type=int, default=0, + help='Index of the central position in the motif (default: 0)') parser.add_argument('--window', type=int, default=8, - help='''Window size around central position. - Total k-mer size is: K = 2*W + 1. (default: 8)''') + help='Window size around central position. Total k-mer size is: K = 2*W + 1 (default: 8)') parser.add_argument('--label', type=int, default=None, help='Label to store for the given examples (default: None)') @@ -267,9 +308,9 @@ def create_arguments() -> argparse.Namespace: # Bisulfite BED file arguments parser.add_argument('--bed_path', type=Path, default=None, help='Path to BED file containing modification information (default: None)') + parser.add_argument('--bed_filter', type=str, default=None, - help='''BED filter method (e.g. high_confidence - finds only high-confidence positions) (default: None)''') + help='BED filter method (e.g. high_confidence finds only high-confidence positions) (default: None)') return parser.parse_args() diff --git a/src/processor/basecall.py b/src/processor/basecall.py index 28ce628..a605111 100644 --- a/src/processor/basecall.py +++ b/src/processor/basecall.py @@ -1,13 +1,14 @@ from pyguppyclient import GuppyBasecallerClient, yield_reads +from pyguppyclient.decode import ReadData, CalledReadData import numpy as np -from typing import List +from typing import Tuple, List from .util import Interval # config='dna_r9.4.1_450bps_hac' for more precise basecalling -def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): +def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast') -> Tuple[ReadData, CalledReadData]: with GuppyBasecallerClient(config_name=config, trace=True) as client: for file in files: for read in yield_reads(file): @@ -15,7 +16,7 @@ def basecall(files: List[str], config: str = 'dna_r9.4.1_450bps_fast'): yield read, called -def sequence_to_raw(read, called) -> List[Interval]: +def sequence_to_raw(read: ReadData, called: CalledReadData) -> List[Interval]: first_signal_id = len(read.signal) - called.trimmed_samples move_index = np.nonzero(called.move)[0] diff --git a/src/processor/custom_processor.py b/src/processor/custom_processor.py index 8b9a3f0..2f5a60d 100644 --- a/src/processor/custom_processor.py +++ b/src/processor/custom_processor.py @@ -1,15 +1,17 @@ +from pyguppyclient.decode import ReadData, CalledReadData import mappy +import numpy as np -from typing import Tuple, Set, Optional +from typing import Tuple, Set, List, Optional from .alignment import make_aligner, get_reference, get_motif_positions from .basecall import sequence_to_raw -from .util import * +from .util import Interval, ResegmentationData class CustomProcessor: def __init__(self, - basecall_data, + basecall_data: Tuple[ReadData, CalledReadData], reference_file: str, mapq: int = 0, motif: str = 'CG', From 4ad96cafc05e1b60a28f3c3a3fe2cc58cf66a30b Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Wed, 2 Jun 2021 04:27:21 +0200 Subject: [PATCH 16/21] Rewrite the BinaryWriter --- src/utils/writer.py | 10 +++++++++- 1 file changed, 9 insertions(+), 1 deletion(-) diff --git a/src/utils/writer.py b/src/utils/writer.py index 1f2f4d0..60fb687 100644 --- a/src/utils/writer.py +++ b/src/utils/writer.py @@ -110,6 +110,7 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None) -> No class BinaryWriter(DataWriter): """ Implementation of DataWriter that stores data in binary file. """ + # TODO remove the hardcoded values TRAIN_DTYPE = np.dtype([('signal', np.float16, (340,)), ('kmer', np.uint8, (17,)), ('label', np.uint8)]) def __init__(self, filename: str) -> None: @@ -132,7 +133,10 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label :param bed_info: Optional BED modification information used for labeling :param label: Optional integer present if label is explicitly given """ + self.fd.write(np.array(len(data.examples)).tobytes()) # Write the number of examples + array = np.empty((len(data.examples),), dtype=BinaryWriter.TRAIN_DTYPE) + no_of_bytes = np.empty((len(data.examples),)) for i, example in enumerate(data.examples): array[i]['signal'] = example.signal_points @@ -147,8 +151,12 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label else: raise ValueError('No label was provided.') + no_of_bytes[i] = len(array[i].tobytes()) + + self.fd.write(no_of_bytes.tobytes()) # Write the number of bytes for every example + array_bytes = array.tobytes() - self.fd.write(array_bytes) + self.fd.write(array_bytes) # Write the examples @staticmethod def on_extraction_finish(*args, **kwargs) -> None: From 3675c65a46a29cdf3bc5ba518f846d06a5b13bdd Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Thu, 10 Jun 2021 01:32:32 +0200 Subject: [PATCH 17/21] Generalize motif positions --- remove-tombo/custom_processor.py | 50 +++++++++++++++++-------------- src/processor/alignment.py | 33 +++++++++++--------- src/processor/custom_processor.py | 13 ++++---- 3 files changed, 55 insertions(+), 41 deletions(-) diff --git a/remove-tombo/custom_processor.py b/remove-tombo/custom_processor.py index 8f98e24..6ce9b67 100644 --- a/remove-tombo/custom_processor.py +++ b/remove-tombo/custom_processor.py @@ -7,7 +7,7 @@ from tqdm import tqdm import numpy as np -from typing import Tuple, Set, List, Optional +from typing import Tuple, Set, List, Dict, Optional from basecall import basecall, sequence_to_raw from util import Interval, ResegmentationData @@ -21,25 +21,30 @@ def make_aligner(reference_file: str) -> mappy.Aligner: return aligner -def get_reference(reference_file: str) -> str: - for seq_record in SeqIO.parse(reference_file, 'fasta'): - reference = str(seq_record.seq) +def get_motif_positions(reference_file: str, motif: str, index: int) -> Dict[str, Tuple[Set[int], Set[int]]]: + chromosomes = SeqIO.to_dict(SeqIO.parse(reference_file, 'fasta')) + motif_positions = dict() - return reference + for chromosome, record in chromosomes.items(): + reference = str(record.seq) + # Forward strand + fwd_matches = re.finditer(motif, reference, re.I) + fwd_pos = set(m.start() + index for m in fwd_matches) -def get_motif_positions(reference: str, motif: str, index: int) -> Tuple[Set[int], Set[int]]: - r_len = len(reference) + # Reverse strand + rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) + rev_pos = set(len(reference) - (m.start() + index) - 1 for m in rev_matches) - # Forward strand - fwd_matches = re.finditer(motif, reference, re.I) - fwd_pos = set(m.start() + index for m in fwd_matches) + motif_positions[chromosome] = fwd_pos, rev_pos - # Reverse strand - rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) - rev_pos = set(r_len - (m.start() + index) - 1 for m in rev_matches) + return motif_positions - return fwd_pos, rev_pos + +def get_reference(reference_file: str, contig: str) -> str: + chromosomes = SeqIO.to_dict(SeqIO.parse(reference_file, 'fasta')) + + return str(chromosomes[contig].seq) def align(aligner: mappy.Aligner, query: str, mapq: int) -> Optional[mappy.Alignment]: @@ -51,10 +56,11 @@ def align(aligner: mappy.Aligner, query: str, mapq: int) -> Optional[mappy.Align return None -def get_relevant_motif_positions(motif_positions: Tuple[Set[int], Set[int]], alignment: mappy.Alignment) -> Set[int]: - strand_pos = motif_positions[0] if alignment.strand == 1 else motif_positions[1] +def get_relevant_motif_positions(motif_positions: Dict[str, Tuple[Set[int], Set[int]]], alignment: mappy.Alignment) -> Set[int]: + contig_positions = motif_positions[alignment.ctg] + strand_positions = contig_positions[0] if alignment.strand == 1 else contig_positions[1] - relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) + relevant_positions = strand_positions & set(range(alignment.r_st, alignment.r_en)) if alignment.strand == 1: return {pos - alignment.r_st for pos in relevant_positions} @@ -139,8 +145,8 @@ def resolve_deletions(signal_intervals: List[Interval], deletion_idx: List[Inter return signal_intervals -def custom_processor(basecall_data: Tuple[ReadData, CalledReadData], aligner: mappy.Aligner, reference: str, - motif_positions: Set[int], mapq: int, window: int) -> ResegmentationData: +def custom_processor(basecall_data: Tuple[ReadData, CalledReadData], aligner: mappy.Aligner, reference_file: str, + motif_positions: Dict[str, Tuple[Set[int], Set[int]]], mapq: int, window: int) -> ResegmentationData: read, called = basecall_data alignment = align(aligner, called.seq, mapq) @@ -168,6 +174,7 @@ def custom_processor(basecall_data: Tuple[ReadData, CalledReadData], aligner: ma event_intervals = signal_intervals[motif_position - window: motif_position + window + 1] event_lens = np.array([interval.end - interval.start for interval in event_intervals]) + reference = get_reference(reference_file, alignment.ctg) region = reference[position - window: position + window + 1] bases = region if alignment.strand == 1 else mappy.revcomp(region) @@ -183,11 +190,10 @@ def process_data(input_path: str, recursive: bool, reference_file: str, fast5_files = get_fast5_files(input_path, recursive=recursive) aligner = make_aligner(reference_file) - reference = get_reference(reference_file) - motif_positions = get_motif_positions(reference, motif, index) + motif_positions = get_motif_positions(reference_file, motif, index) for basecall_data in tqdm(basecall(fast5_files)): - resegmentation_data = custom_processor(basecall_data, aligner, reference, motif_positions, mapq, window) + resegmentation_data = custom_processor(basecall_data, aligner, reference_file, motif_positions, mapq, window) if resegmentation_data: print(resegmentation_data) diff --git a/src/processor/alignment.py b/src/processor/alignment.py index 230d065..7811c82 100644 --- a/src/processor/alignment.py +++ b/src/processor/alignment.py @@ -2,7 +2,7 @@ from Bio import SeqIO import re -from typing import Tuple, Set +from typing import Tuple, Set, Dict def make_aligner(reference_file: str) -> mappy.Aligner: @@ -13,22 +13,27 @@ def make_aligner(reference_file: str) -> mappy.Aligner: return aligner -def get_reference(reference_file: str) -> str: - for seq_record in SeqIO.parse(reference_file, 'fasta'): - reference = str(seq_record.seq) +def get_motif_positions(reference_file: str, motif: str, index: int) -> Dict[str, Tuple[Set[int], Set[int]]]: + chromosomes = SeqIO.to_dict(SeqIO.parse(reference_file, 'fasta')) + motif_positions = dict() - return reference + for chromosome, record in chromosomes.items(): + reference = str(record.seq) + # Forward strand + fwd_matches = re.finditer(motif, reference, re.I) + fwd_pos = set(m.start() + index for m in fwd_matches) -def get_motif_positions(reference: str, motif: str, index: int) -> Tuple[Set[int], Set[int]]: - r_len = len(reference) + # Reverse strand + rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) + rev_pos = set(len(reference) - (m.start() + index) - 1 for m in rev_matches) - # Forward strand - fwd_matches = re.finditer(motif, reference, re.I) - fwd_pos = set(m.start() + index for m in fwd_matches) + motif_positions[chromosome] = fwd_pos, rev_pos - # Reverse strand - rev_matches = re.finditer(motif, mappy.revcomp(reference), re.I) - rev_pos = set(r_len - (m.start() + index) - 1 for m in rev_matches) + return motif_positions - return fwd_pos, rev_pos \ No newline at end of file + +def get_reference(reference_file: str, contig: str) -> str: + chromosomes = SeqIO.to_dict(SeqIO.parse(reference_file, 'fasta')) + + return str(chromosomes[contig].seq) diff --git a/src/processor/custom_processor.py b/src/processor/custom_processor.py index 2f5a60d..49866f4 100644 --- a/src/processor/custom_processor.py +++ b/src/processor/custom_processor.py @@ -18,12 +18,13 @@ def __init__(self, index: int = 0, window: int = 8): self.basecall_data = basecall_data + self.reference_file = reference_file + self.mapq = mapq self.window = window self.aligner = make_aligner(reference_file) - self.reference = get_reference(reference_file) - self.motif_positions = get_motif_positions(self.reference, motif, index) + self.motif_positions = get_motif_positions(reference_file, motif, index) def align(self, query: str) -> Optional[mappy.Alignment]: for hit in self.aligner.map(query): # Traverse alignments @@ -34,9 +35,10 @@ def align(self, query: str) -> Optional[mappy.Alignment]: return None def _get_relevant_motif_positions(self, alignment: mappy.Alignment) -> Set[int]: - strand_pos = self.motif_positions[0] if alignment.strand == 1 else self.motif_positions[1] + contig_positions = self.motif_positions[alignment.ctg] + strand_positions = contig_positions[0] if alignment.strand == 1 else contig_positions[1] - relevant_positions = strand_pos & set(range(alignment.r_st, alignment.r_en)) + relevant_positions = strand_positions & set(range(alignment.r_st, alignment.r_en)) if alignment.strand == 1: return {pos - alignment.r_st for pos in relevant_positions} @@ -148,7 +150,8 @@ def process(self) -> Optional[List[ResegmentationData]]: event_intervals = signal_intervals[motif_position - self.window: motif_position + self.window + 1] event_lens = np.array([interval.end - interval.start for interval in event_intervals]) - region = self.reference[position - self.window: position + self.window + 1] + reference = get_reference(self.reference_file, alignment.ctg) + region = reference[position - self.window: position + self.window + 1] bases = region if alignment.strand == 1 else mappy.revcomp(region) assert len(event_intervals) == len(event_lens) == len(bases) From 542207cc5de96e8133b4f4cc57e31bf4a0e3bde3 Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Thu, 10 Jun 2021 17:31:58 +0200 Subject: [PATCH 18/21] Rewrite the code for signals of variable lengths --- src/extract_features.py | 20 ++++---- src/utils/datasets.py | 108 +++++++++++++++++++++++----------------- src/utils/models.py | 3 +- src/utils/writer.py | 57 +++++++++++++-------- 4 files changed, 110 insertions(+), 78 deletions(-) diff --git a/src/extract_features.py b/src/extract_features.py index 0abe351..ed8589e 100644 --- a/src/extract_features.py +++ b/src/extract_features.py @@ -104,7 +104,7 @@ def generate_data(read: ReadData, example_points = np.concatenate(example_points) - example = Example(reseg_example.position, reseg_example.bases, example_points) + example = Example(reseg_example.position, reseg_example.bases, example_points, reseg_example.event_lens) all_examples.append(example) if len(all_examples) == 0: @@ -178,17 +178,18 @@ def init_workers( _BED_DATA = bed_data -def worker_process_reads(paths: List[Path], reference: str, out_path: Path) -> Tuple[Path, int]: +def worker_process_reads(paths: List[Path], reference: str, data_path: Path, header_path: Path) -> Tuple[Path, int]: """ Function that processes input file list and stores generated data :param paths: List of input files that will be processed :param reference: Path to the reference file - :param out_path: Path to the generated output + :param data_path: Path to the generated output data + :param header_path: Path to the generated header :return: Path and number of failed files """ error_files = 0 - with BinaryWriter(str(out_path)) as writer: + with BinaryWriter(str(data_path), str(header_path)) as writer: bed_pos = set(_BED_DATA.keys()) if _BED_DATA is not None else None for path in tqdm(basecall(paths)): @@ -198,10 +199,10 @@ def worker_process_reads(paths: List[Path], reference: str, out_path: Path) -> T if result is not None: writer.write_data(result, _BED_DATA, _INFO_ARGS['label']) except Exception as e: - # error_callback(path, e) - error_files += 1 + error_callback(path, e) + # error_files += 1 - return out_path, error_files + return data_path, error_files def tqdm_with_time(msg, last_action_time): @@ -251,9 +252,10 @@ def process_data(args: argparse.Namespace) -> None: start = i * BATCH_SIZE end = min(start+BATCH_SIZE, len(files)) - out_file_path = Path(args.output_path, f'{i+1}.bin.tmp') + data_path = Path(args.output_path, f'{i+1}.data.bin.tmp') + header_path = Path(args.output_path, f'{i+1}.header.bin.tmp') - future = executor.submit(worker_process_reads, files[start:end], args.reference, out_file_path) + future = executor.submit(worker_process_reads, files[start:end], args.reference, data_path, header_path) futures.append(future) tqdm_with_time('Extracting features', last_action_time) diff --git a/src/utils/datasets.py b/src/utils/datasets.py index b89711b..5194f95 100644 --- a/src/utils/datasets.py +++ b/src/utils/datasets.py @@ -12,14 +12,14 @@ from tqdm import tqdm -DATA_DTYPE = np.dtype([('signal', np.float16, (340,)), ('kmer', np.uint8, (17,)), ('label', np.uint8)]) +# DATA_DTYPE = np.dtype([('signal', np.float16, (340,)), ('kmer', np.uint8, (17,)), ('label', np.uint8)]) def dataset_size(path): - length, r = divmod(os.path.getsize(path), DATA_DTYPE.itemsize) - assert r == 0, 'Cannot calculate a number of examples. Possibly wrong data file.' + with io.open(path.replace('data.bin', 'header.bin'), 'rb') as f: + signal = np.fromfile(f, dtype=np.uint16) - return length + return len(signal) def different_length_error(filename, read_name): @@ -29,49 +29,65 @@ def different_length_error(filename, read_name): class MemoryDataset(Dataset): def __init__(self, path): - with io.open(path, 'rb') as f: - self.data = np.fromfile(f, dtype=DATA_DTYPE) + self.data = io.open(path, 'rb') - def __len__(self): - return len(self.data) - - def __getitem__(self, idx): - example = self.data[idx] - - signal = torch.from_numpy(example['signal']) - bases = torch.from_numpy(np.repeat(example['kmer'], 20).astype(int)) - label = example['label'] - - return signal, bases, label + with open(path.replace('data.bin', 'info.txt')) as f: + window = int(f.readlines()[4].split()[1]) + self.kmer = window * 2 + 1 + with io.open(path.replace('data.bin', 'header.bin'), 'rb') as f: + self.signal = np.fromfile(f, dtype=np.uint16) -def iter_worker_init(worker_id): - worker_info = torch.utils.data.get_worker_info() - dataset = worker_info.dataset - - dataset.f = io.open(dataset.path, 'rb') - - -class IterDataset(Dataset): - def __init__(self, path): - self.len = dataset_size(path) - - self.path = path + self.example_bytes = self.signal * 2 + self.kmer * 3 + 1 # Lengths of examples in bytes + self.offset = np.concatenate(([0], self.example_bytes)).cumsum()[:-1] def __len__(self): - return self.len + return len(self.signal) def __getitem__(self, idx): - self.f.seek(idx * DATA_DTYPE.itemsize) - example = np.frombuffer(self.f.read(DATA_DTYPE.itemsize), dtype=DATA_DTYPE)[0] + data_dtype = np.dtype([('signal', np.float16, (self.signal[idx],)), + ('lens', np.uint16, (self.kmer,)), + ('kmer', np.uint8, (self.kmer,)), + ('label', np.uint8)]) + + self.data.seek(self.offset[idx]) + example = np.frombuffer(self.data.read(self.example_bytes[idx]), data_dtype)[0] signal = torch.from_numpy(example['signal']) - bases = torch.from_numpy(np.repeat(example['kmer'], 20).astype(int)) + lens = example['lens'] + bases = torch.from_numpy(np.repeat(example['kmer'], lens).astype(int)) label = example['label'] return signal, bases, label +# def iter_worker_init(worker_id): +# worker_info = torch.utils.data.get_worker_info() +# dataset = worker_info.dataset +# +# dataset.f = io.open(dataset.path, 'rb') +# +# +# class IterDataset(Dataset): +# def __init__(self, path): +# self.len = dataset_size(path) +# +# self.path = path +# +# def __len__(self): +# return self.len +# +# def __getitem__(self, idx): +# self.f.seek(idx * DATA_DTYPE.itemsize) +# example = np.frombuffer(self.f.read(DATA_DTYPE.itemsize), dtype=DATA_DTYPE)[0] +# +# signal = torch.from_numpy(example['signal']) +# bases = torch.from_numpy(np.repeat(example['kmer'], 20).astype(int)) +# label = example['label'] +# +# return signal, bases, label + + class RockfishDataModule(LightningDataModule): def __init__(self, train_path, val_path, train_batch_size, val_batch_size, iterable): super().__init__() @@ -88,25 +104,25 @@ def __init__(self, train_path, val_path, train_batch_size, val_batch_size, itera def train_dataloader(self): worker_init_fn = None - if self.iterable: - train_ds = IterDataset(self.train_path) - worker_init_fn = iter_worker_init - else: - train_ds = MemoryDataset(self.train_path) + # if self.iterable: + # train_ds = IterDataset(self.train_path) + # worker_init_fn = iter_worker_init + # else: + train_ds = MemoryDataset(self.train_path) return DataLoader(train_ds, self.train_batch_size, True, - num_workers=4, pin_memory=True, drop_last=True, worker_init_fn=worker_init_fn) + num_workers=4, pin_memory=True, drop_last=True, worker_init_fn=worker_init_fn) def val_dataloader(self): worker_init_fn = None - if self.iterable: - val_ds = IterDataset(self.val_path) - worker_init_fn = iter_worker_init - else: - val_ds = MemoryDataset(self.val_path) + # if self.iterable: + # val_ds = IterDataset(self.val_path) + # worker_init_fn = iter_worker_init + # else: + val_ds = MemoryDataset(self.val_path) return DataLoader(val_ds, self.val_batch_size, num_workers=4, - pin_memory=True, worker_init_fn=worker_init_fn) + pin_memory=True, worker_init_fn=worker_init_fn) @classmethod def add_argparse_args(cls, parent_parser): @@ -124,4 +140,4 @@ def add_argparse_args(cls, parent_parser): @classmethod def from_argparse_args(cls, args): return RockfishDataModule(args.train_path, args.val_path, - args.train_batch_size, args.val_batch_size, args.iterable) + args.train_batch_size, args.val_batch_size, args.iterable) diff --git a/src/utils/models.py b/src/utils/models.py index 721dd54..6c795c3 100644 --- a/src/utils/models.py +++ b/src/utils/models.py @@ -446,11 +446,12 @@ class Example: """ Data class that contains information about one example. This data class stores information about example's position on the reference genome, reference k-mer around the - position and sampled signal points. + position, sampled signal points and lengths of intervals of signal points. """ position: int ref_kmer: str signal_points: np.ndarray + event_lens: np.ndarray """@dataclass diff --git a/src/utils/writer.py b/src/utils/writer.py index 60fb687..b43199d 100644 --- a/src/utils/writer.py +++ b/src/utils/writer.py @@ -110,21 +110,22 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None) -> No class BinaryWriter(DataWriter): """ Implementation of DataWriter that stores data in binary file. """ - # TODO remove the hardcoded values - TRAIN_DTYPE = np.dtype([('signal', np.float16, (340,)), ('kmer', np.uint8, (17,)), ('label', np.uint8)]) - - def __init__(self, filename: str) -> None: + def __init__(self, data_filename: str, header_filename: str) -> None: super().__init__() - self.filename = filename + self.data_filename = data_filename + self.header_filename = header_filename def __enter__(self): - self.fd = io.open(self.filename, 'wb') + self.data_fd = io.open(self.data_filename, 'wb') + self.header_fd = io.open(self.header_filename, 'wb') return self def __exit__(self, type, value, traceback): - self.fd.flush() - self.fd.close() + self.data_fd.flush() + self.data_fd.close() + self.header_fd.flush() + self.header_fd.close() def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label: Optional[int]=None) -> None: """ Function that writes data into binary file. @@ -133,39 +134,51 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label :param bed_info: Optional BED modification information used for labeling :param label: Optional integer present if label is explicitly given """ - self.fd.write(np.array(len(data.examples)).tobytes()) # Write the number of examples + no_of_signals = [] + + for example in data.examples: + data_dtype = np.dtype([('signal', np.float16, (len(example.signal_points),)), + ('lens', np.uint16, (len(example.event_lens),)), + ('kmer', np.uint8, (len(example.ref_kmer),)), + ('label', np.uint8)]) - array = np.empty((len(data.examples),), dtype=BinaryWriter.TRAIN_DTYPE) - no_of_bytes = np.empty((len(data.examples),)) + array = np.empty(1, dtype=data_dtype) - for i, example in enumerate(data.examples): - array[i]['signal'] = example.signal_points - array[i]['kmer'] = [base_encoding[base] for base in example.ref_kmer] + array[0]['signal'] = example.signal_points + array[0]['lens'] = example.event_lens + array[0]['kmer'] = [base_encoding[base] for base in example.ref_kmer] if bed_info is not None: mod_info = bed_info[GenomicPos(data.chromosome, example.position, data.strand)] bed_label = 1 if mod_info.mod_freq > 50 else 0 - array[i]['label'] = bed_label + array[0]['label'] = bed_label elif label is not None: - array[i]['label'] = label + array[0]['label'] = label else: raise ValueError('No label was provided.') - no_of_bytes[i] = len(array[i].tobytes()) + array_bytes = array.tobytes() + no_of_signals.append(len(array[0]['signal'])) - self.fd.write(no_of_bytes.tobytes()) # Write the number of bytes for every example + self.data_fd.write(array_bytes) - array_bytes = array.tobytes() - self.fd.write(array_bytes) # Write the examples + self.header_fd.write(np.array(no_of_signals, dtype=np.uint16).tobytes()) @staticmethod def on_extraction_finish(*args, **kwargs) -> None: - """ Function that concatenates temporary files into a single output and removes temporary files. """ + """Function that concatenates temporary files into a single output and removes temporary files.""" - src_path = Path(kwargs['path'], '*.bin.tmp') + src_path = Path(kwargs['path'], '*.data.bin.tmp') dest_path = Path(kwargs['path'], 'data.bin') cat_command = f'cat {src_path} > {dest_path}' + subprocess.run(cat_command, shell=True) + rm_command = f'rm {src_path}' + subprocess.run(rm_command, shell=True) + + src_path = Path(kwargs['path'], '*.header.bin.tmp') + dest_path = Path(kwargs['path'], 'header.bin') + cat_command = f'cat {src_path} > {dest_path}' subprocess.run(cat_command, shell=True) rm_command = f'rm {src_path}' From 77c6f54b571794d4d89f54a9dbc19c5cdd389c5d Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Thu, 10 Jun 2021 20:13:29 +0200 Subject: [PATCH 19/21] Concatenate header and data files, code enhancement --- src/utils/datasets.py | 27 ++++++++++++++------------- src/utils/writer.py | 37 +++++++++++++++++++++++++------------ 2 files changed, 39 insertions(+), 25 deletions(-) diff --git a/src/utils/datasets.py b/src/utils/datasets.py index 5194f95..1a285a4 100644 --- a/src/utils/datasets.py +++ b/src/utils/datasets.py @@ -13,13 +13,15 @@ # DATA_DTYPE = np.dtype([('signal', np.float16, (340,)), ('kmer', np.uint8, (17,)), ('label', np.uint8)]) +NUM_16 = 2 # 2 bytes +NUM_8 = 1 # 1 byte def dataset_size(path): - with io.open(path.replace('data.bin', 'header.bin'), 'rb') as f: - signal = np.fromfile(f, dtype=np.uint16) + with io.open(path, 'rb') as f: + number_of_examples = np.frombuffer(f.read(NUM_16), dtype=np.uint16)[0] - return len(signal) + return number_of_examples def different_length_error(filename, read_name): @@ -30,19 +32,18 @@ def different_length_error(filename, read_name): class MemoryDataset(Dataset): def __init__(self, path): self.data = io.open(path, 'rb') + self.len = dataset_size(path) # Number of examples with open(path.replace('data.bin', 'info.txt')) as f: window = int(f.readlines()[4].split()[1]) - self.kmer = window * 2 + 1 + self.kmer = 2 * window + 1 # Length of kmer - with io.open(path.replace('data.bin', 'header.bin'), 'rb') as f: - self.signal = np.fromfile(f, dtype=np.uint16) - - self.example_bytes = self.signal * 2 + self.kmer * 3 + 1 # Lengths of examples in bytes - self.offset = np.concatenate(([0], self.example_bytes)).cumsum()[:-1] + self.signal = np.frombuffer(self.data.read(NUM_16 * self.len), dtype=np.uint16) # Lengths of signals + example_bytes = NUM_16 * self.signal + (NUM_16 + NUM_8) * self.kmer + NUM_8 # Lengths of examples in bytes + self.offset = np.concatenate(([0], example_bytes)).cumsum()[:-1] # Offsets in bytes def __len__(self): - return len(self.signal) + return self.len def __getitem__(self, idx): data_dtype = np.dtype([('signal', np.float16, (self.signal[idx],)), @@ -50,10 +51,10 @@ def __getitem__(self, idx): ('kmer', np.uint8, (self.kmer,)), ('label', np.uint8)]) - self.data.seek(self.offset[idx]) - example = np.frombuffer(self.data.read(self.example_bytes[idx]), data_dtype)[0] + self.data.seek(NUM_16 + NUM_16 * self.len + self.offset[idx]) + example = np.frombuffer(self.data.read(data_dtype.itemsize), data_dtype)[0] - signal = torch.from_numpy(example['signal']) + signal = torch.from_numpy(example['signal'].astype(np.float16)) lens = example['lens'] bases = torch.from_numpy(np.repeat(example['kmer'], lens).astype(int)) label = example['label'] diff --git a/src/utils/writer.py b/src/utils/writer.py index b43199d..9b4e0a1 100644 --- a/src/utils/writer.py +++ b/src/utils/writer.py @@ -134,7 +134,7 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label :param bed_info: Optional BED modification information used for labeling :param label: Optional integer present if label is explicitly given """ - no_of_signals = [] + signal_lengths = [] for example in data.examples: data_dtype = np.dtype([('signal', np.float16, (len(example.signal_points),)), @@ -158,28 +158,41 @@ def write_data(self, data: FeaturesData, bed_info: Optional[BEDData]=None, label raise ValueError('No label was provided.') array_bytes = array.tobytes() - no_of_signals.append(len(array[0]['signal'])) + signal_lengths.append(len(array[0]['signal'])) self.data_fd.write(array_bytes) - self.header_fd.write(np.array(no_of_signals, dtype=np.uint16).tobytes()) + self.header_fd.write(np.array(signal_lengths, dtype=np.uint16).tobytes()) @staticmethod def on_extraction_finish(*args, **kwargs) -> None: """Function that concatenates temporary files into a single output and removes temporary files.""" - src_path = Path(kwargs['path'], '*.data.bin.tmp') - dest_path = Path(kwargs['path'], 'data.bin') - cat_command = f'cat {src_path} > {dest_path}' + # Concatenate temporary data files + src_data_path = Path(kwargs['path'], '*.data.bin.tmp') + dest_data_path = Path(kwargs['path'], 'data_no_header.bin') + cat_command = f'cat {src_data_path} > {dest_data_path}' subprocess.run(cat_command, shell=True) - rm_command = f'rm {src_path}' - subprocess.run(rm_command, shell=True) + # Concatenate temporary header files + src_header_path = Path(kwargs['path'], '*.header.bin.tmp') + dest_header_path = Path(kwargs['path'], 'header.bin') + cat_command = f'cat {src_header_path} > {dest_header_path}' + subprocess.run(cat_command, shell=True) - src_path = Path(kwargs['path'], '*.header.bin.tmp') - dest_path = Path(kwargs['path'], 'header.bin') - cat_command = f'cat {src_path} > {dest_path}' + # Add number of examples to the beginning of header file + with io.open(dest_header_path, 'rb+') as f: + header = np.fromfile(f, dtype=np.uint16) + no_of_examples = len(header) + header = np.concatenate(([no_of_examples], header)) + f.seek(0) + f.write(header.astype('uint16').tobytes()) + + # Concatenate header and data file to a final data.bin file + final_data_path = Path(kwargs['path'], 'data.bin') + cat_command = f'cat {dest_header_path} {dest_data_path} > {final_data_path}' subprocess.run(cat_command, shell=True) - rm_command = f'rm {src_path}' + # Delete unnecessary files + rm_command = f'rm {src_data_path} {src_header_path} {dest_data_path} {dest_header_path}' subprocess.run(rm_command, shell=True) From ab5a9e7d8fbc3937376e0f547014cf81069f248e Mon Sep 17 00:00:00 2001 From: Sanja Deur Date: Fri, 11 Jun 2021 06:27:37 +0200 Subject: [PATCH 20/21] Added histograms --- remove-tombo/plots/ratio_D_mod_final.png | Bin 0 -> 24655 bytes remove-tombo/plots/ratio_D_nomod_final.png | Bin 0 -> 24993 bytes remove-tombo/plots/signal_points_D.png | Bin 0 -> 26901 bytes remove-tombo/plots/signal_points_M.png | Bin 0 -> 24097 bytes remove-tombo/scripts/plot_signal_points.py | 47 ++++++++++++++++++ .../scripts/plot_signal_points_ratios.py | 38 ++++++++++++++ 6 files changed, 85 insertions(+) create mode 100644 remove-tombo/plots/ratio_D_mod_final.png create mode 100644 remove-tombo/plots/ratio_D_nomod_final.png create mode 100644 remove-tombo/plots/signal_points_D.png create mode 100644 remove-tombo/plots/signal_points_M.png 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