Bulk Fastq Quantification

Overview

OmicVerse provides a complete bulk RNA-seq FASTQ-to-count-matrix pipeline via the ov.alignment module. This skill covers:

• SRA data acquisition: prefetch and fqdump (fasterq-dump wrapper), or parallel_fastq_dump (multi-threaded .lite.1 → paired FASTQ)
• Quality control: fastp for adapter trimming and QC reports
• Path A — alignment-based: STAR aligner with auto-index building + featureCount (subread featureCounts wrapper) for gene quantification. Produces sorted BAMs and a gene × sample count DataFrame. Use when transcript structure / splicing matters or when downstream tools expect BAMs.
• Path B — alignment-free (kb-python technology='BULK'): ov.alignment.single.ref builds a kallisto index + t2g + cDNA once per genome; ov.alignment.count(technology='BULK', ...) quantifies each sample directly to gene counts. Much faster than STAR + featureCounts on the same machine, with comparable gene-level accuracy for most DE use cases. This is the t_mapping_kbpython walkthrough.
• DE handoff: wiring per-sample outputs into ov.bulk.pyDEG (DESeq2 / edgeR / limma) → volcano plot.

All functions share a common CLI infrastructure (_cli_utils.py) that handles tool resolution, auto-installation via conda/mamba, parallel execution, and streaming output.

Out of scope: single-cell kb-python (technology='10XV2', '10XV3', velocity workflows). See single-cell-kb-alignment for that.

Instructions

1. Environment setup

   • Bioinformatics tools are resolved automatically from PATH or the active conda environment.
   • If auto_install=True (default), missing tools are installed via mamba/conda on demand.
   • Supported tools for Path A: prefetch, vdb-validate, fasterq-dump, fastp, STAR, samtools, featureCounts, pigz, gzip.
   • For Path B (kb-python): pip install kb-python.

2. SRA data download (ov.alignment.prefetch + ov.alignment.fqdump, or ov.alignment.parallel_fastq_dump)

   • Use prefetch first for reliable downloads with integrity validation (vdb-validate).
   • Then convert to FASTQ with fqdump. It auto-detects single-end vs paired-end.
   • fqdump can also work directly from SRR accessions without prefetch.
   • For .lite.1 direct downloads (e.g. NCBI mirrors), use ov.datasets.download_data(url, dir=...) + ov.alignment.parallel_fastq_dump instead.
   • All variants support retry with exponential backoff for network errors.

   import omicverse as ov
   
   # Variant 1 — SRR accession via prefetch + fqdump
   pre = ov.alignment.prefetch(['SRR1234567', 'SRR1234568'],
                                output_dir='prefetch', jobs=4)
   fq  = ov.alignment.fqdump(['SRR1234567', 'SRR1234568'],
                              output_dir='fastq', sra_dir='prefetch',
                              gzip=True, threads=8, jobs=4)
   
   # Variant 2 — direct .lite.1 link + parallel_fastq_dump (paired-end)
   ov.datasets.download_data(
       'https://sra-downloadb.be-md.ncbi.nlm.nih.gov/.../SRR12544419.lite.1',
       dir='./data',
   )
   ov.alignment.parallel_fastq_dump(
       sra_id='./data/SRR12544419.lite.1',
       outdir='./data/SRR12544419',
       tmpdir='./tmp',
       threads=12, split_files=True, gzip=True,
   )
   

3. FASTQ quality control (ov.alignment.fastp)

   • Runs fastp for adapter trimming, quality filtering, and QC reporting.
   • Supports single-end and paired-end reads.
   • Produces per-sample JSON and HTML QC reports.
   • Sample format: tuple of (sample_name, fq1_path, fq2_path_or_None).
   • Optional for Path B — kb-python can consume raw FASTQs directly, but fastp still improves DE quality on noisy SRA inputs.

   samples = [
       ('S1', 'fastq/SRR1234567/SRR1234567_1.fastq.gz', 'fastq/SRR1234567/SRR1234567_2.fastq.gz'),
       ('S2', 'fastq/SRR1234568/SRR1234568_1.fastq.gz', 'fastq/SRR1234568/SRR1234568_2.fastq.gz'),
   ]
   clean = ov.alignment.fastp(samples, output_dir='fastp', threads=8, jobs=2)
   

Path A — STAR + featureCounts (alignment-based)

4. STAR alignment (ov.alignment.STAR)

   • Aligns FASTQ reads using the STAR aligner.
   • Auto-index building: set auto_index=True (default) with genome_fasta_files and gtf to build index automatically if missing.
   • Produces coordinate-sorted BAM files.
   • Handles gzip-compressed FASTQs automatically (uses pigz/gzip/zcat).
   • Use strict=False (default) for graceful error handling per sample.

   star_samples = [
       ('S1', 'fastp/S1/S1_clean_1.fastq.gz', 'fastp/S1/S1_clean_2.fastq.gz'),
       ('S2', 'fastp/S2/S2_clean_1.fastq.gz', 'fastp/S2/S2_clean_2.fastq.gz'),
   ]
   bams = ov.alignment.STAR(
       star_samples,
       genome_dir='star_index',
       output_dir='star_out',
       gtf='genes.gtf',
       genome_fasta_files=['genome.fa'],
       threads=8,
       memory='50G',
   )
   

5. Gene quantification (ov.alignment.featureCount)

   • Counts aligned reads per gene using featureCounts (subread).
   • Auto-detects paired-end from BAM headers (via pysam or samtools).
   • auto_fix=True (default) retries with corrected paired-end flag on error.
   • gene_mapping=True maps gene_id to gene_name from the GTF.
   • merge_matrix=True produces a combined count matrix across all samples.

   bam_items = [
       ('S1', 'star_out/S1/Aligned.sortedByCoord.out.bam'),
       ('S2', 'star_out/S2/Aligned.sortedByCoord.out.bam'),
   ]
   counts = ov.alignment.featureCount(
       bam_items,
       gtf='genes.gtf',
       output_dir='counts',
       gene_mapping=True,
       merge_matrix=True,
       threads=8,
   )
   # counts is a pandas DataFrame (gene_id × samples) — feed directly to pyDEG
   

6. Path A wiring

   • fastp → STAR: fastp output is a list of dicts with keys sample, clean1, clean2, json, html.

     star_samples = [
         (r['sample'], r['clean1'], r['clean2'] if r['clean2'] else None)
         for r in (clean if isinstance(clean, list) else [clean])
     ]
     

   • STAR → featureCount: STAR output is a list of dicts with keys sample, bam (or error).

     bam_items = [
         (r['sample'], r['bam'])
         for r in (bams if isinstance(bams, list) else [bams])
         if 'bam' in r
     ]
     

Path B — kb-python technology='BULK' (alignment-free)

7. Build the kb reference (ov.alignment.single.ref)

   • Builds the kallisto index, the transcript-to-gene map (t2g.txt), and the transcriptome FASTA (cdna.fa). Once per genome+annotation; reuse across the whole cohort.
   • Keep FASTA and GTF from the same Ensembl release to avoid transcript-ID mismatches.

   ref_result = ov.alignment.single.ref(
       fasta_paths='genomes/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz',
       gtf_paths='genomes/Homo_sapiens.GRCh38.108.gtf.gz',
       index_path='kb_ref/index.idx',
       t2g_path='kb_ref/t2g.txt',
       cdna_path='kb_ref/cdna.fa',
       temp_dir='tmp',
       overwrite=False,
   )
   

8. Quantify each bulk sample (ov.alignment.count with technology='BULK')

   • technology='BULK' disables barcode/UMI handling — one observation per sample.
   • Required bulk-specific args: filter_barcodes=False, parity='paired'|'single', strand='unstranded'|'forward'|'reverse'. The wrapper raises if technology='BULK' is combined with filter_barcodes=True.
   • Output per sample: <output_path>/counts_unfiltered/adata.h5ad (when h5ad=True) plus cells_x_genes.genes.names.txt.

   for sra in ['SRR12544419', 'SRR12544421', 'SRR12544433', 'SRR12544435']:
       ov.alignment.count(
           fastq_paths=[
               f'./data/{sra}/{sra}.lite.1_1.fastq.gz',
               f'./data/{sra}/{sra}.lite.1_2.fastq.gz',
           ],
           index_path='kb_ref/index.idx',
           t2g_path='kb_ref/t2g.txt',
           technology='BULK',
           output_path=f'results/{sra}/',
           h5ad=True,
           filter_barcodes=False,
           parity='paired',
           strand='unstranded',
           threads=12,
       )
   

Handoff to differential expression

9. Wiring bulk outputs into ov.bulk.pyDEG (DESeq2)

   Both paths end with a gene × sample integer count matrix that pyDEG consumes.

   From Path A — already a pandas DataFrame:

   dds = ov.bulk.pyDEG(counts)         # counts from ov.alignment.featureCount(..., merge_matrix=True)
   

   From Path B — load per-sample h5ad, harmonise gene names, concatenate, transpose:

   ad_dict = {}
   for sra in ['SRR12544419', 'SRR12544421', 'SRR12544433', 'SRR12544435']:
       ad = ov.read(f'./results/{sra}/counts_unfiltered/adata.h5ad')
       gene_name = ov.pd.read_csv(
           f'./results/{sra}/counts_unfiltered/cells_x_genes.genes.names.txt',
           header=None,
       )
       ad.var['gene_name'] = gene_name[0].tolist()
       ad.var['gene_id']   = ad.var.index
       ad.var.index        = ad.var['gene_name']
       ad.var_names_make_unique()
       ad.obs['sra']       = sra
       ad_dict[sra] = ad
   
   adata = ov.concat(ad_dict)
   adata.obs_names_make_unique()
   adata.obs['Group'] = ['no', 'no', 'yes', 'yes']   # phenotype labels
   
   counts = adata.to_df().T                          # gene × sample
   dds = ov.bulk.pyDEG(counts)
   

   Run DE + filter + volcano (same regardless of upstream path):

   dds.drop_duplicates_index()
   result = dds.deg_analysis(
       treatment_groups=[...],   # sample IDs in the treatment arm (matrix columns)
       control_groups=[...],     # sample IDs in the control arm
       method='DEseq2',
   )
   # Optional: filter low-expression genes after DE
   result = result.loc[result['log2(BaseMean)'] > 1]
   
   dds.foldchange_set(fc_threshold=-1, pval_threshold=0.05, logp_max=10)
   dds.plot_volcano(title='DEG Analysis', figsize=(4, 4),
                    plot_genes_num=8, plot_genes_fontsize=12)
   

   • DESeq2 expects raw integer counts — do not log-normalize before deg_analysis.
   • treatment_groups / control_groups are sample IDs (matrix columns), not group labels.
   • For downstream GSEA / pathway / PPI, see the gsea-enrichment, bulk-deg-analysis, bulk-stringdb-ppi skills.

10. Skipping completed steps

    • All functions check for existing outputs and skip if overwrite=False (default).
    • Set overwrite=True to force re-execution.

11. Troubleshooting

    • If a tool is not found, check auto_install=True and that conda/mamba is accessible.
    • For STAR index errors, ensure genome_fasta_files points to uncompressed or gzip FASTA files.
    • For featureCounts paired-end detection errors, auto_fix=True handles most cases automatically.
    • GTF files can be gzip-compressed; they are auto-decompressed as needed.
    • For kb-python BULK: if quantification returns zero counts, double-check parity matches the actual library (single vs paired) and that strand matches the kit (most modern Illumina mRNA-seq is unstranded or reverse).

Critical API Reference

Sample format convention (Path A)

All alignment-based functions use a consistent sample tuple format:

• FASTQ samples: (sample_name, fq1_path, fq2_path_or_None)
• BAM items: (sample_name, bam_path) or (sample_name, bam_path, is_paired_bool)
• Single samples can be passed as a single tuple; multiple as a list of tuples.
• When a single tuple is passed, the return value is a single dict; for a list, a list of dicts.

Auto-installation

# All functions support these parameters:
auto_install=True   # Auto-install missing tools via conda/mamba
overwrite=False     # Skip if outputs already exist
threads=8           # Per-tool thread count
jobs=None           # Concurrent job count (auto-detected from CPU count)

Examples

• Bulk via STAR (alignment-based): prefetch → fqdump → fastp → STAR → featureCount → DataFrame → ov.bulk.pyDEG DESeq2 → volcano. (t_mapping_STAR)
• Bulk via kb-python (alignment-free): download_data → parallel_fastq_dump → ov.alignment.single.ref → ov.alignment.count(technology='BULK', filter_barcodes=False, parity='paired', strand='unstranded') → per-sample h5ad → ov.concat → ov.bulk.pyDEG DESeq2 → volcano. (t_mapping_kbpython)
• Local FASTQ + kb-python: skip download, jump straight to ov.alignment.single.ref + ov.alignment.count(technology='BULK', ...).
• Local FASTQ + STAR: skip download, start at fastp → STAR → featureCount.

Related skills

• single-cell-kb-alignment — single-cell kb-python path (technology='10XV2' / '10XV3' / velocity workflows). Same ov.alignment.single.ref + ov.alignment.count API; different technology and barcode/UMI handling.
• bulk-deseq2-analysis — pure DESeq2 differential-expression analysis on an existing count matrix.
• bulk-deg-analysis, gsea-enrichment, bulk-stringdb-ppi — downstream DE/enrichment/network skills.

References

• See reference.md for copy-paste-ready code templates.
• Tutorial notebooks:
  • t_mapping_STAR.ipynb — STAR + featureCounts walkthrough.
  • t_mapping_kbpython.ipynb — kb-python bulk walkthrough with DESeq2.