3. Bulk RNAseq

Quality control

Read alignment using HISAT2

Building HISAT2 index

cd /path/to/genome_dir
mkdir -p Hisat2_index

# The name of the index files will be as genome.x.ht# and will be saved in the Hisat2_index directory
hisat2-build -p 38 genome.fa Hisat2_index/genome


# Generate file with known splice junctions to be fed to HISAT2
hisat2_extract_exons.py genome.gtf > genome_known_splice_sites.txt

Aligning Paired-end reads using HISAT2

cd /PATH/TO/PROJECT/outputs
mkdir -p Hisat2_SAMs



# parse fastq data into arrays
for fwd in PATH/TO/PROJECT/originals/FASTQ/*.R1.fastq.gz;do
   rev=${fwd/R1/R2}    # Change the pattern and replacement accordingly
   name=$( basename $fwd )
   name=${name%.R1.fastq.gz}  # Change pattern accordingly
   hisat2 -p 20 --dta --known-splicesite-infile /path/to/genome_known_splice_sites.txt -x /path/to/Hisat2_index/genome \
     -1 $fwd -2 $rev -S Hisat2_SAMs/${name}.sam --no-discordant
done

For Single-end reads, the same code can be used, but replace -1 with -U and exclude the pair read.

Converting SAMs to BAMs

Because HISAT outputs SAM files, we will have to convert to a more portable BAM format.

# sort and convert .sam files into BAM format
cd /PATH/TO/PROJECTS/output
mkdir Hisat2_sorted_BAMs

for sam in Hisat2_SAMs/*.sam; do
    name=$( basename $sam )
    name=${name%.sam}
    samtools view -@ 20 -Su $sam | samtools sort -@ 20 -o Hisat2_sorted_BAMs/$name.bam
done

# index bam files
cd Hisat2_sorted_BAMs
for filename in *.bam; do
    samtools index -@ 40 $filename $filename.bai
done

Construct custom transcriptome

We use StringTie2 to construct our own custom transcriptome containing de novo transcripts.

cd /PATH/TO/PROJECTS/output
mkdir -p Stringtie_gtf

for bam in Hisat2_sorted_BAMs/*.bam; do
	name=$( basename $bam )
        name=${name%.bam}
	stringtie $bam -p 20 -o Stringtie_gtf/$name.gtf -G /path/to/referenceGTF
done

Transcript quantiification using Kallisto

Splicing analyses using Whippet

Intron retention analysis using VAST-Tools

Intron retention analysis using IRFinder