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NGS data analysis — four pipelines

Coursework projects from the NGS data analysis course of the MSc in Data Analysis in Biology and Medicine, HSE University (2025). Each folder holds the code and a write-up of what was done, what came out, and what I would do differently.

Project Data Stack Question
01 — Bulk RNA-seq 6 single-end libraries, wine flor yeast, day 10 vs day 71 fastp, HISAT2, featureCounts, DESeq2, kallisto What changes in the transcriptome as a flor biofilm ages?
02 — scRNA-seq Human nucleus accumbens, 10x v3, single-nuclei scanpy, Scrublet, UMAP From raw matrix to annotated cell types, without mistaking depth for biology
03 — 16S amplicon 60 paired-end samples, gut microbiome under TB preventive therapy DADA2, SILVA 138, phyloseq Reproducing a published microbiome analysis end to end
04 — Exome variant calling WES BAM, coronary artery disease case Picard, DeepVariant, bcftools, SnpEff, ClinVar Which variants could a clinician act on?

What these projects show

  • Full pipelines, not single steps — raw reads through QC, alignment or denoising, quantification, statistics, and biological interpretation.
  • QC treated as evidence. Read attrition is accounted for stage by stage; an overrepresented sequence is BLASTed rather than ignored; mean–variance behaviour is inspected before and after normalisation.
  • Batch and design structure taken seriously — donor effects in the brain atlas, treatment groups in the microbiome study, capture regions in the exome.
  • Honest limits. Where a constraint broke an analysis (see the subsampling mistake in project 03), it is written down with the lesson, not smoothed over.

Tools

fastp · Trimmomatic · Trim Galore · FastQC / MultiQC · HISAT2 · samtools · featureCounts · kallisto · DESeq2 · scanpy · Scrublet · DADA2 · phyloseq · SILVA · Picard · DeepVariant · bcftools · SnpEff · ClinVar — in Python, R and bash, with Docker where a tool needs it.

Note on the material

Input data are not redistributed here: they are course-supplied files or public archives (SRA PRJNA772261, CELLxGENE, the DeepVariant exome case-study bucket), and each project README says where its inputs come from. The assignment texts themselves are course material and are not included.

The code and analyses in this repository are mine.

Author: Appolinaria Prokopovich — GitHub · LinkedIn

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End-to-end NGS analyses - bulk and single-cell RNA-seq, 16s microbiome, exome variant calling - with QC treated as evidence and limitation written down

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