Bulk RNA-seq Data Standards – PanKbase

This document describes data generation, processing, and QC standards in PanKbase for bulk RNA-sequencing studies, largely adopted from standards developed by the GTEx consortium.

Overview

Bulk RNA-sequencing (RNA-seq) produces genomic sequencing data describing the abundance of RNA transcripts in a biosample from RNA molecules such as protein-coding and long non-coding RNA transcripts.

I. Data Generation

Data generation standards (from GTEx):

• Read length: At least 50 bp
• Insert size: Average library insert size should be 200 base pairs
• Sequencing depth: Minimum of 15 million reads per RNA-seq library
• Replicates:
  • Two or more biological replicates
    • Anisogenic: Biosamples from different donors
    • Isogenic: Independent biosamples from the same donor
  • Technical replicates (optional): Replicate experiments or replicate sequencing of the same biosample

> Although not required, RNA-seq experiments could include quantitative standards such as spike-ins of RNA of known length and quantity to help calibrate quantification, sensitivity, and coverage.

II. Data Processing

The recommended pipeline is the GTEx RNA-seq pipeline.

GTEx v10 Pipeline:

• Software versions:
  • STAR v2.7.10a
  • RSEM v1.3.3
  • RNA-SeQC v2.4.2
• Resources:
  • Human genome: GRCh38
  • Annotation: GENCODE v39

Key pipeline steps:

1. Building indexes:
   • STAR and RSEM indexes must be built specifically for each read length
2. Alignment:
   • STAR aligner is used to map sequence reads to transcripts
3. Mark duplicates:
   • PICARD tools are used to mark duplicate reads in alignments
4. Quality control:
   • RNA-SeQC is used for quality control of alignments
5. Quantification:
   • RSEM is used to quantify transcripts

Pipeline Outputs

• Genome alignment: .bam
• Transcript alignment: .bam
• Normalized signal: .bigWig
• Gene quantifications: .tsv
• Transcript quantifications: .tsv
• Quality control metrics: .txt

III. Data Quality Control

High-quality samples are defined based on GTEx criteria:

• Aligned reads:
  • >10 million aligned reads (or mate-pairs if paired-end)
• Mapping rate criteria:
  • Read mapping rate > 0.2
  • Intergenic mapping rate < 0.3
  • Base mismatch rate < 0.01 for mate 1 or < 0.02 for mate 2
  • Ribosomal RNA (rRNA) mapping rate < 0.3

Recommended correlations:

• Spearman correlation > 0.9 between isogenic replicates (same donor)
• Spearman correlation > 0.8 between anisogenic replicates (different donors within the same study)