Problem with the Parsnp step

[Vincev454]

Hello,

Thank you for developing one of the few tools that computes strain abundance, this has been very helpful!

I ran into one issue during the Parsnp step (I used a pair of fastq file from Nextseq sequencing as a test input);

Main error message:
`[Tue Dec 16 06:15:20 2025]
localrule run_parsnp:
input: /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000820765.1_Arquibacterium_massiliensis_AP7_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_004216775.1_ASM421677v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_013009555.1_ASM1300955v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_018292205.1_ASM1829220v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_020554845.1_ASM2055484v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_040267485.1_UoG_LM19B_1.0_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_041014885.1_Genome_assembly_GPTB0036_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042850115.1_ASM4285011v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042854215.1_ASM4285421v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_044591705.1_ASM4459170v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_045061075.1_ASM4506107v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046111155.1_Anaerostipes_caccae_LAHUC_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603875.1_ASM4660387v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603935.1_ASM4660393v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_048192685.1_ASM4819268v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_050366995.1_ASM5036699v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051904065.1_ASM5190406v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051905015.1_ASM5190501v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052348685.1_ASM5234868v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052377865.1_ASM5237786v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052880695.1_ASM5288069v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna
output: /home/vince5/scratch/Strainify/Results_out/parsnp_results/parsnp.maf
jobid: 28
reason: Missing output files: /home/vince5/scratch/Strainify/Results_out/parsnp_results/parsnp.maf; Input files updated by another job: /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042854215.1_ASM4285421v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042850115.1_ASM4285011v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_050366995.1_ASM5036699v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603875.1_ASM4660387v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_020554845.1_ASM2055484v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_040267485.1_UoG_LM19B_1.0_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046111155.1_Anaerostipes_caccae_LAHUC_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052880695.1_ASM5288069v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051905015.1_ASM5190501v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_013009555.1_ASM1300955v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_041014885.1_Genome_assembly_GPTB0036_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603935.1_ASM4660393v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052377865.1_ASM5237786v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_004216775.1_ASM421677v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_044591705.1_ASM4459170v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_018292205.1_ASM1829220v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000820765.1_Arquibacterium_massiliensis_AP7_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051904065.1_ASM5190406v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_048192685.1_ASM4819268v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_045061075.1_ASM4506107v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052348685.1_ASM5234868v1_genomic.fna
threads: 12
resources: tmpdir=/tmp

06:15:21 - ^[[1;37mINFO^[[0m - |--Parsnp 2.1.4--|

06:15:21 - ^[[1;33mWARNING^[[0m - Output directory /home/vince5/scratch/Strainify/Results_out/parsnp_results exists, all results will be overwritten
06:15:21 - ^[[1;37mINFO^[[0m -

---

SETTINGS:
|-refgenome: autopick
|-genomes:
/home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna
/home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna
...21 more file(s)...
/home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna
/home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna
|-aligner: muscle
|-outdir: /home/vince5/scratch/Strainify/Results_out/parsnp_results
|-OS: Linux
|-threads: 12

---

06:15:21 - ^[[1;37mINFO^[[0m - <>
06:15:21 - ^[[1;37mINFO^[[0m - No genbank file provided for reference annotations, skipping..
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna is 5.49x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna is 1.78x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000820765.1_Arquibacterium_massiliensis_AP7_genomic.fna is 2.30x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_004216775.1_ASM421677v1_genomic.fna is 1.98x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_013009555.1_ASM1300955v1_genomic.fna is 1.22x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_018292205.1_ASM1829220v1_genomic.fna is 1.25x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_020554845.1_ASM2055484v1_genomic.fna is 1.67x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_040267485.1_UoG_LM19B_1.0_genomic.fna is 1.30x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042850115.1_ASM4285011v1_genomic.fna is 1.45x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042854215.1_ASM4285421v1_genomic.fna is 1.82x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_044591705.1_ASM4459170v1_genomic.fna is 2.30x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_045061075.1_ASM4506107v1_genomic.fna is 2.13x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046111155.1_Anaerostipes_caccae_LAHUC_genomic.fna is 1.97x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603875.1_ASM4660387v1_genomic.fna is 1.76x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_048192685.1_ASM4819268v1_genomic.fna is 2.03x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_050366995.1_ASM5036699v1_genomic.fna is 1.46x shorter than reference genome!
06:15:21 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051904065.1_ASM5190406v1_genomic.fna is 1.33x shorter than reference genome!
06:15:22 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051905015.1_ASM5190501v1_genomic.fna is 1.25x shorter than reference genome!
06:15:22 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052377865.1_ASM5237786v1_genomic.fna is 2.58x shorter than reference genome!
06:15:22 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052880695.1_ASM5288069v1_genomic.fna is 5.49x shorter than reference genome!
06:15:22 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna is 1.56x shorter than reference genome!
06:15:22 - ^[[1;33mWARNING^[[0m - File /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna is 1.24x shorter than reference genome!
06:15:22 - ^[[1;37mINFO^[[0m - Too few genomes to run partitions of size >50. Running all genomes at once.
06:15:22 - ^[[1;37mINFO^[[0m - Running Parsnp multi-MUM search and libMUSCLE aligner...
06:16:14 - ^[[1;31mCRITICAL^[[0m - Set of filtered genomes are too divergent for parsnp, please use stricter ANI thresholds for filtering
06:16:14 - ^[[1;31mCRITICAL^[[0m - Aligned regions cover less than 1% of reference genome, something is not right

Adjust params and rerun. If issue persists please submit a GitHub issue

RuleException:
CalledProcessError in file "/scratch/vince5/Strainify/Snakefile", line 105:
Command 'set -euo pipefail;
mkdir -p /home/vince5/scratch/Strainify/Results_out/parsnp_results
parsnp -r ! -o /home/vince5/scratch/Strainify/Results_out/parsnp_results -c -p 12 --fo -d /home/vince5/scratch/Strainify/Results_out/renamed_genomes' returned non-zero exit status 1.
[Tue Dec 16 06:16:14 2025]
Error in rule run_parsnp:
message: None
jobid: 28
input: /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000820765.1_Arquibacterium_massiliensis_AP7_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_004216775.1_ASM421677v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_013009555.1_ASM1300955v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_018292205.1_ASM1829220v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_020554845.1_ASM2055484v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_040267485.1_UoG_LM19B_1.0_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_041014885.1_Genome_assembly_GPTB0036_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042850115.1_ASM4285011v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042854215.1_ASM4285421v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_044591705.1_ASM4459170v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_045061075.1_ASM4506107v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046111155.1_Anaerostipes_caccae_LAHUC_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603875.1_ASM4660387v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603935.1_ASM4660393v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_048192685.1_ASM4819268v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_050366995.1_ASM5036699v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051904065.1_ASM5190406v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051905015.1_ASM5190501v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052348685.1_ASM5234868v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052377865.1_ASM5237786v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052880695.1_ASM5288069v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna
output: /home/vince5/scratch/Strainify/Results_out/parsnp_results/parsnp.maf
shell:

        mkdir -p /home/vince5/scratch/Strainify/Results_out/parsnp_results
        parsnp -r ! -o /home/vince5/scratch/Strainify/Results_out/parsnp_results -c -p 12 --fo -d /home/vince5/scratch/Strainify/Results_out/renamed_genomes

    (command exited with non-zero exit code)

Shutting down, this might take some time.
Exiting because a job execution failed. Look below for error messages
[Tue Dec 16 06:16:16 2025]
Error in rule run_parsnp:
message: None
jobid: 28
input: /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCA_050873045.1_ASM5087304v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000269565.1_PB_Copr_sp_D6_V1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_000820765.1_Arquibacterium_massiliensis_AP7_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_004216775.1_ASM421677v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_013009555.1_ASM1300955v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_018292205.1_ASM1829220v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_020554845.1_ASM2055484v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_040267485.1_UoG_LM19B_1.0_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_041014885.1_Genome_assembly_GPTB0036_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042850115.1_ASM4285011v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_042854215.1_ASM4285421v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_044591705.1_ASM4459170v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_045061075.1_ASM4506107v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046111155.1_Anaerostipes_caccae_LAHUC_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603875.1_ASM4660387v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_046603935.1_ASM4660393v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_048192685.1_ASM4819268v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_050366995.1_ASM5036699v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051904065.1_ASM5190406v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_051905015.1_ASM5190501v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052348685.1_ASM5234868v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052377865.1_ASM5237786v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_052880695.1_ASM5288069v1_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_900445495.1_52683_B04_genomic.fna, /home/vince5/scratch/Strainify/Results_out/renamed_genomes/GCF_964259335.1_CC00595_genomic.fna
output: /home/vince5/scratch/Strainify/Results_out/parsnp_results/parsnp.maf
shell:

        mkdir -p /home/vince5/scratch/Strainify/Results_out/parsnp_results
        parsnp -r ! -o /home/vince5/scratch/Strainify/Results_out/parsnp_results -c -p 12 --fo -d /home/vince5/scratch/Strainify/Results_out/renamed_genomes

    (command exited with non-zero exit code)

Complete log(s): /scratch/vince5/Strainify/.snakemake/log/2025-12-16T061517.167396.snakemake.log
WorkflowError:
At least one job did not complete successfully.`

Could you please tell me how I can solve it?

Many thanks,
Vince

[rossie-luo]

Hi Vince,

Thank you for reaching out! I see that your input genomes are quite different in length. Since the Parsnp flag is set to "-c" (the current default), Parsnp failed at producing output. You could try adding this to your config.yaml file: parsnp_flags: --skip-ani-filter. This should keep the maximum number of genomes that can be aligned (the ones that are either much longer or shorter than the rest of the genomes will be kicked out). Let me know if you have any other questions!

Best,
Rossie

[Vincev454]

Hi Rossie,

Thanks a lot for your help!
However, I got exactly the same error as above when using parsnp_flags: --skip-ani-filter. Should I look for other FASTA sequences with more homogeneous lengths for my reference genomes?

Best,
Vince

[rossie-luo]

Hi Vince,

Sorry for the late reply! Any chance you could share your FASTA files with me so that I could look into the Parsnp output?

Thanks,
Rossie

[Vincev454]

Hi Rossie,

No worries! Here is a link to the reference genomes:
https://drive.google.com/file/d/1r3KUa6kySklvMhkz7KdyrPgT6mFEvX9Q/view?usp=drive_link

Thank you for your help and best wishes for 2026!
Vince

[rossie-luo]

Hi Vince,

I ran your genomes but it looks like Parsnp did not find any MUMs (identical sequences in the genomes used as anchors for alignment). This means the genomes are too divergent and no core genome was found. Are these genomes from the same species?

Best,
Rossie

[Vincev454]

Hi Rossie,

My mistake was indeed that silly: I mixed up the reference genomes, which in fact did not belong to the same species. Sorry for the wasted time, and thank you for your help.

Best,
Vince

[rossie-luo]

No worries at all, happy to help! Let me know if you have other questions.