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How to analyze RNA-seq data with hisat2

This recipe performs an RNA Seq analysis of spiked control samples using an alignment based method

1 result • updated 2.1 years ago by Bioinformatics Admin

This recipe performs an RNA Seq analysis of spiked control samples using an alignment based method

For more information see the

Bioinformatics Data Analysis online course.
Biostar Handbook for system setup and other information.

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# Print the commands as are executed.
# Stop on errors.
set -uex

# The URL the data is located at.
URL=http://data.biostarhandbook.com/rnaseq/projects/griffith/griffith-data.tar.gz

# Obtain and download the data
wget -q -nc $URL

# Unpack the dataset.
tar zxvf griffith-data.tar.gz

# This is the reference genome.
REF=refs/22.fa

# Represents the annotations.
GTF=refs/22.gtf

# Build a hisat2 index for the genome.
hisat2-build $REF $REF 2>> log.txt 1>> log.txt

# Generate the root names for the files.
parallel -j 1 echo {1}_{2} ::: UHR HBR ::: 1 2 3 > names.txt

# Make a directory for BAM files.
mkdir -p bam

# Run the alignment per each root name.
cat names.txt | parallel "hisat2 $REF -1 reads/{}_R1.fq -2 reads/{}_R2.fq | samtools sort > bam/{}.bam" 2>> log.txt

# Generate a BAM index for each BAm file.
cat names.txt | parallel "samtools index bam/{}.bam" 2>> log.txt

# Count the features that overlap annotations.
featureCounts -p -a $GTF -g gene_name -o counts.txt  bam/*.bam 2>> log.txt

# Get the script that runs the deseq 1 method.
wget -q -nc  http://data.biostarhandbook.com/rnaseq/code/deseq1.r

# Simplify the count matrix to only contain gene names and read counts.
# Cut out only the columns that the differential expression script expects.
cat counts.txt | cut -f 1,7-12 > simple_counts.txt

# Perform the differential expression study.
cat simple_counts.txt | Rscript deseq1.r 3x3 > results.txt 2>> log.txt

# Download the script to draw the heatmaps (requires the glplots package).
wget -q -nc http://data.biostarhandbook.com/rnaseq/code/draw-heatmap.r

# Generate heatmap from the deseq1 normalized matrix created by the deseq1.r script.
cat norm-matrix-deseq1.txt | Rscript draw-heatmap.r > heatmap-norm-matrix-deseq1.pdf  2>> log.txt

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