A. Genome-based quantification (STAR + featureCounts)

Last updated on 2025-11-25 | Edit this page

Estimated time: 70 minutes

Overview

Questions

How do we map RNA-seq reads to a reference genome?
How do we determine library strandness before alignment?
How do we quantify reads per gene using featureCounts?
How do we submit mapping jobs on RCAC clusters with SLURM?

Objectives

Check RNA-seq library strandness using Salmon.
Index a reference genome for STAR.
Map reads to the genome using STAR.
Quantify gene level counts with featureCounts.
Interpret basic alignment and counting statistics.

Introduction

In this episode we move from raw FASTQ files to genome based gene counts.
This workflow has three major steps:

Determine library strandness: STAR does not infer strandness automatically. featureCounts requires the correct setting.
Map reads to the genome using STAR: STAR is a splice aware aligner designed for RNA-seq data.
Count mapped reads per gene using featureCounts

This represents one of the two standard quantification strategies in RNA-seq.
The alternative method (B. transcript based quantification with Salmon or Kallisto) will be covered in Episode 4B.

Step 1: Checking RNA-seq library strandness

Callout

Why strandness matters

RNA-seq libraries can be:

unstranded
stranded forward
stranded reverse

Stranded protocols are common in modern kits, but old GEO and SRA datasets are often unstranded.
featureCounts must be given the correct strand setting. If the wrong setting is used, most reads will be counted against incorrect genes.

STAR does not infer strandness automatically, so we must determine it before alignment.

Detecting strandness with Salmon

Salmon can infer strandness directly from raw FASTQ files without any alignment.
We run Salmon with -l A, which tells it to explore all library types.

BASH

sinteractive -A workshop -q standby -p cpu -N 1 -n 4 --time=1:00:00

cd $SCRATCH/rnaseq-workshop
mkdir -p results/strand_check

module load biocontainers
module load salmon

salmon index --transcripts data/gencode.vM38.transcripts-clean.fa \
             --index data/salmon_index \
             --threads 4

salmon quant --index data/salmon_index \
             --libType A \
             --mates1 data/SRR33253286_1.fastq.gz \
             --mates2 data/SRR33253286_2.fastq.gz \
             --output results/strand_check \
             --threads 4

The important output is in results/strand_check/lib_format_counts.json:

What does this file look like?

JSON

{
    "read_files": "[ data/SRR33253286_1.fastq.gz, data/SRR33253286_2.fastq.gz]",
    "expected_format": "IU",
    "compatible_fragment_ratio": 1.0,
    "num_compatible_fragments": 21088696,
    "num_assigned_fragments": 21088696,
    "num_frags_with_concordant_consistent_mappings": 19116702,
    "num_frags_with_inconsistent_or_orphan_mappings": 2510542,
    "strand_mapping_bias": 0.5117980078362889,
    "MSF": 0,
    "OSF": 0,
    "ISF": 9783890,
    "MSR": 0,
    "OSR": 0,
    "ISR": 9332812,
    "SF": 1234245,
    "SR": 1276297,
    "MU": 0,
    "OU": 0,
    "IU": 0,
    "U": 0
}

Interpreting lib_format_counts.json

This JSON file reports Salmon’s automatic library type detection.

"expected_format": "IU": This is Salmon’s conclusion. “I” means Inward (correct for paired-end reads) and “U” means Unstranded.

"strand_mapping_bias": 0.512: This is the key evidence. A value near 0.5 (50%) indicates that reads mapped equally to both the sense and antisense strands, the definitive sign of an unstranded library.

"ISF" and "ISR": These are the counts for Inward-Sense-Forward (9.78M) and Inward-Sense-Reverse (9.33M) fragments. Because these values are almost equal, they confirm the ~50/50 split seen in the strand bias.

Common results:

Code	Meaning
IU	unstranded
ISR	stranded reverse (paired end)
ISF	stranded forward (paired end)
SR	stranded reverse (single end)
SF	stranded forward (single end)

Conclusion: The data is unstranded. We will record this and supply it to STAR/featureCounts.

Step 2: Preparing the reference genome for alignment

STAR requires a genome index. Indexing builds a searchable data structure that speeds up alignment.

Required input:

genome FASTA file
annotation file (GTF or GFF3)
output directory for the index

Optionally, annotation improves splice junction detection and yields better alignments.

Callout

Why STAR?

Splice-aware
Extremely fast
Produces high-quality alignments
Well supported and widely used
Compatible with downstream tools such as featureCounts, StringTie, and RSEM

Optional alternatives include HISAT2 and Bowtie2, but STAR remains the standard for high-depth Illumina RNA-seq.

Challenge

Exercise: Read the STAR manual

Check the “Generating genome indexes” section of the
STAR manual

Which options are required for indexing?

Show me the solution

Key options:

--runMode genomeGenerate
--genomeDir directory to store the index
--genomeFastaFiles the FASTA file
--sjdbGTFfile annotation (recommended)
--runThreadN number of threads
--sjdbOverhang read length minus one (100 is a safe default)

Indexing the Genome with STAR

Below is a minimal SLURM job script for building the STAR genome index. Create a file named index_genome.sh in $SCRATCH/rcac_rnaseq/scripts with this content:

BASH

#!/bin/bash
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --cpus-per-task=20
#SBATCH --account=workshop
#SBATCH --partition=cpu
#SBATCH --time=1:00:00
#SBATCH --job-name=star_index
#SBATCH --output=cluster-%x.%j.out
#SBATCH --error=cluster-%x.%j.err

module load biocontainers
module load star

data_dir="${SCRATCH}/rnaseq-workshop/data"
genome="${data_dir}/GRCm39.primary_assembly.genome.fa"
gtf="${data_dir}/gencode.vM38.primary_assembly.basic.annotation.gtf"
indexdir="${data_dir}/star_index"

STAR \
    --runMode genomeGenerate \
    --runThreadN ${SLURM_CPUS_ON_NODE} \
    --genomeDir ${indexdir} \
    --genomeFastaFiles ${genome} \
    --sjdbGTFfile ${gtf}

Submit the job:

BASH

sbatch index_genome.sh

Indexing requires about 30 to 45 minutes.

What will the output look like?

The index directory is now ready for use in mapping and should have contents like this: Location: $SCRATCH/rcac_rnaseq/data Contents of star_index/:

BASH

star_index/
├── chrLength.txt
├── chrNameLength.txt
├── chrName.txt
├── chrStart.txt
├── exonGeTrInfo.tab
├── exonInfo.tab
├── geneInfo.tab
├── Genome
├── genomeParameters.txt
├── Log.out
├── SA
├── SAindex
├── sjdbInfo.txt
├── sjdbList.fromGTF.out.tab
├── sjdbList.out.tab
└── transcriptInfo.tab

Callout

Do I need the GTF for indexing?

No, but it is strongly recommended.
Including it improves splice junction detection and increases mapping accuracy.

Step 3: Mapping reads with STAR

Once the genome index is ready, we can align reads.

For aligning reads, important STAR options are:

--runThreadN
--genomeDir
--readFilesIn
--readFilesCommand zcat for gzipped FASTQ files
--outSAMtype BAM SortedByCoordinate
--outSAMunmapped Within

STAR’s defaults are tuned for mammalian genomes. If working with plants, fungi, or repetitive genomes, additional tuning may be required.

Challenge

Exercise: Write a STAR alignment command

Write a basic STAR alignment command using your genome index and paired FASTQ files.

Show me the solution

BASH

STAR \
    --runThreadN 12 \
    --genomeDir data/star_index \
    --readFilesIn data/SRR1234567_1.fastq.gz data/SRR1234567_2.fastq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix results/mapping/SRR1234567 \
    --outSAMtype BAM SortedByCoordinate \
    --outSAMunmapped Within

Mapping many FASTQ files with a SLURM array

When having multiple samples, array jobs simplify submission.

First, we will create samples.txt file with just the sample names

BASH

cd $SCRATCH/rnaseq-workshop/data
ls *_1.fastq.gz | sed 's/_1.fastq.gz//' > ${SCRATCH}/rnaseq-workshop/scripts/samples.txt

BASH

#!/bin/bash
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --cpus-per-task=20
#SBATCH --account=workshop
#SBATCH --partition=cpu
#SBATCH --time=8:00:00
#SBATCH --job-name=read_mapping
#SBATCH --array=1-6
#SBATCH --output=cluster-%x.%j.out
#SBATCH --error=cluster-%x.%j.err

module load biocontainers
module load star

FASTQ_DIR="$SCRATCH/rnaseq-workshop/data"
GENOME_INDEX="$SCRATCH/rnaseq-workshop/data/star_index"
OUTDIR="$SCRATCH/rnaseq-workshop/results/mapping"

mkdir -p $OUTDIR

SAMPLE=$(sed -n "${SLURM_ARRAY_TASK_ID}p" samples.txt)

R1=${FASTQ_DIR}/${SAMPLE}_1.fastq.gz
R2=${FASTQ_DIR}/${SAMPLE}_2.fastq.gz

STAR \
    --runThreadN ${SLURM_CPUS_ON_NODE} \
    --genomeDir $GENOME_INDEX \
    --readFilesIn $R1 $R2 \
    --readFilesCommand zcat \
    --outFileNamePrefix $OUTDIR/$SAMPLE \
    --outSAMunmapped Within \
    --outSAMtype BAM SortedByCoordinate

Submit:

BASH

sbatch map_reads.sh

Discussion

Why use an array job?

Think about this:

We have six samples, each with two FASTQ files
Having six separate slurm jobs means:
- Copy-pasting the same command six times
- Higher chance of typos or mistakes
if mapping with single slurm file, sequential execution = slower
Samples can run independently

Why is a job array a good choice?

Arrays allow us to run identical commands across many inputs:

All samples are processed the same way
Runs in parallel
Simplifies submission
Reduces copy-paste errors
Minimizes runtime cost on the cluster

Step 4: Assessing alignment quality

STAR produces several useful files, particularly:

Log.final.out
Contains mapping statistics, including % uniquely mapped.
Aligned.sortedByCoord.out.bam
Ready for downstream quantification.

To summarize alignment statistics across samples, we use MultiQC:

BASH

cd $SCRATCH/rnaseq-workshop
module load biocontainers
module load multiqc

multiqc results/mapping -o results/qc_alignment

Key metrics to review

Uniquely mapped reads — typically 60–90% is good
Multi-mapped reads — depends on genome, but >20% is concerning
Unmapped reads — check reasons (mismatches? too short?)
Mismatch rate — high values indicate quality or contamination issues

Challenge

Exercise: Examine your alignment metrics

Using your MultiQC alignment report:

Which sample has the highest uniquely mapped percentage?
Are any samples clear outliers?
Does mapped/unmapped reads appear consistent across samples?

Show me the solution

Example interpretation for this dataset:

All samples have ~80% uniquely mapped reads.
No sample is an outlier.
mapped/unmapped reads consistent across samples

Step 5: Quantifying gene counts with featureCounts

Once the reads have been aligned and sorted, we can quantify how many read pairs map to each gene. This gives us gene-level counts that we will later use for differential expression. For alignment-based RNA-seq workflows, featureCounts is the recommended tool because it is fast, robust, and compatible with most gene annotation formats.

featureCounts uses two inputs:

A set of aligned BAM files (sorted by coordinate)
A GTF annotation file describing gene models

It assigns each read (or read pair) to a gene based on the exon boundaries.

Callout

Important: stranded or unstranded?

featureCounts needs to know whether your dataset is stranded.

-s 0 unstranded
-s 1 stranded forward
-s 2 stranded reverse

From our Salmon strandness check, this dataset behaves as unstranded, so we will use -s 0.

Running featureCounts

We will create a new directory for count output:

BASH

cd $SCRATCH/rnaseq-workshop/scripts
mkdir -p $SCRATCH/rnaseq-workshop/results/counts

Below is an example SLURM script to count all BAM files at once.

BASH

#!/bin/bash
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --cpus-per-task=16
#SBATCH --account=workshop
#SBATCH --partition=cpu
#SBATCH --time=1:00:00
#SBATCH --job-name=featurecounts
#SBATCH --output=cluster-%x.%j.out
#SBATCH --error=cluster-%x.%j.err

module load biocontainers
module load subread

GTF="$SCRATCH/rnaseq-workshop/data/gencode.vM38.annotation.gtf"
BAMS="$SCRATCH/rnaseq-workshop/results/mapping/*.bam"
output_dir="$SCRATCH/rnaseq-workshop/results/counts"

featureCounts \
  -T ${SLURM_CPUS_PER_TASK} \
  -p \
  -s 0 \
  -t exon \
  -g gene_id \
  -a ${GTF} \
  -o ${output_dir}/gene_counts.txt \
  ${BAMS}

Submit the job:

BASH

sbatch count_features.sh

gene_counts.txt will contain:

one row per gene
one column per sample

Callout

Generate a clean count matrix

BASH

cd $SCRATCH/rnaseq-workshop/results/counts
cut -f 1,7- gene_counts.txt |\
  sed 's+/scratch/negishi/'$USER'/rnaseq-workshop/results/mapping/++g' |\
  grep -v "^#" |\
  sed 's/Aligned.sortedByCoord.out.bam//g' \
  > gene_counts_clean.txt

Step 6: QC on counts

Before proceeding to differential expression, it is good practice to perform some QC on the count data. We can again use MultiQC to summarize featureCounts statistics:

BASH

cd $SCRATCH/rnaseq-workshop
mkdir -p results/qc_counts
module load biocontainers
module load multiqc
multiqc results/counts -o results/qc_counts

Challenge

Exercise: Examine your counting metrics

Using your MultiQC featureCounts report:

Which sample has the lowest percent assigned, and what are common reasons for lower assignment?
Which unassigned category is the largest, and what does it indicate?
Does the sample with the most assigned reads also have the highest percent assigned?
What does “Unassigned: No Features” mean, and why might it occur?
Are multi-mapped reads a concern for RNA-seq differential expression?

Show me the solution

Example interpretation for this dataset:

SRR33253289 has the lowest assignment rate. Causes include low complexity, incomplete annotation, or more intronic reads.
Unmapped or No Features dominate. These reflect reads that do not align or do not overlap annotated exons.
No. Total assigned reads depend on depth, while percent assigned reflects library quality.
Reads mapped outside annotated exons, often from intronic regions, unannotated transcripts, or incomplete annotation.
Multi-mapping is expected for paralogs and repeats. It is usually fine as long as the rate is consistent across samples.

Summary

Key Points

STAR is a fast splice-aware aligner widely used for RNA-seq.
Genome indexing requires FASTA and optionally GTF for improved splice detection.
Mapping is efficiently performed using SLURM array jobs.
Alignment statistics (from Log.final.out + MultiQC) must be reviewed.
Strandness should be inferred using aligned BAM files.
Final BAM files are ready for downstream counting.
featureCounts is used to obtain gene-level counts for differential expression.
Exons are counted and summed per gene.
The output is a simple matrix for downstream statistical analysis.

A. Genome-based quantification (STAR + featureCounts)

Overview

Questions

Objectives

Introduction

Step 1: Checking RNA-seq library strandness

Why strandness matters

Detecting strandness with Salmon

BASH

What does this file look like?

JSON

Interpreting lib_format_counts.json

Step 2: Preparing the reference genome for alignment

Why STAR?

Exercise: Read the STAR manual

Show me the solution

Indexing the Genome with STAR

BASH

BASH

What will the output look like?

BASH

Do I need the GTF for indexing?

Step 3: Mapping reads with STAR

Exercise: Write a STAR alignment command

Show me the solution

BASH

Mapping many FASTQ files with a SLURM array

BASH

BASH

BASH

Why use an array job?

Step 4: Assessing alignment quality

BASH

Key metrics to review

Exercise: Examine your alignment metrics

Show me the solution

Step 5: Quantifying gene counts with featureCounts

Important: stranded or unstranded?

Running featureCounts

BASH

BASH

BASH

Generate a clean count matrix

BASH

Step 6: QC on counts

BASH

Exercise: Examine your counting metrics

Show me the solution

Summary

Interpreting `lib_format_counts.json`