NGS-Methods-Development

A repository for next-generation sequencing (NGS) methods development and benchmarking projects. This repository contains analyses comparing different library preparation approaches and custom protocol optimizations.

Projects

1. scRNAComparison

Comparison of Single-Cell RNA-Seq Library Preparation Kits

This project benchmarks different commercially available plate-based single-cell RNA-seq library preparation kits against published reference data.

• Objective: Evaluate the quality and accuracy of datasets generated from different commercial scRNA-seq prep kits
• Approach: Compared 4 commercial kits (48 cells each) with published data from Li et al. (GEO: GSE117233, 288 cells)
• Outputs: Clustering analysis, quality metrics, and comparative visualizations

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2. TestCustomOligosForDrosophilaRiboDepletion

Custom Ribosomal Depletion Oligos for Drosophila RNA-Seq

This project tests custom-designed oligonucleotides for ribosomal RNA depletion in Drosophila melanogaster samples, optimized for use with the KAPA RiboErase kit.

• Objective: Develop and validate species-specific ribo-depletion oligos for fly samples
• Approach: Designed custom oligos targeting 28S, 18S, 16S, 5S, and 5.8S rRNA; tested with/without 16S oligos (due to AT-rich content)
• Conditions tested:
  • Human-Mouse-Rat oligos (standard kit control)
  • Custom fly oligos with 16S depletion
  • Custom fly oligos without 16S depletion
• Key findings: Custom fly oligos dramatically reduced rRNA contamination (~0.2-2% vs ~65-73% with HMR oligos)

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Repository Structure

NGS-Methods-Development/
├── README.md
├── scRNAComparison/
│   ├── README.md
│   └── scRNA-Comp.R
└── TestCustomOligosForDrosophilaRiboDepletion/
    ├── README.md
    ├── clustering.png
    └── scatterplots-and-corr.jpeg

License

This project is available for academic and research use.