Reference SOP / 06
Bulk RNA-seq: alignment workflow.
This reference workflow covers standard gene-level quantification from paired-end reads. The project plan should define the reference build, strandedness, biological contrasts, and exclusion criteria before analysis begins.
Core sequence
WORKING REFERENCE
FASTQ + sample sheet
fastp
HISAT2 index
HISAT2 alignment
QC + counts
The minimum archive includes the sample sheet, reference FASTA and annotation identifiers, fastp reports, HISAT2 and samtools statistics, a gene-count matrix, software versions, parameters, logs, and the script that joins sample-level results.
- Validate sample names, read pairs, checksums, and metadata before processing.
- Run fastp with adapter detection and quality filtering; retain both HTML and JSON reports.
- Build the HISAT2 index once from the declared reference; store the command, annotation splice sites, and reference checksum.
- Align each sample with a consistent strandedness policy; sort and index BAM files and capture alignment statistics.
- Quantify genes with a declared annotation and counting rule; verify library complexity, mapping rate, duplication, and sample relationships before differential analysis.