Ctrl+K

Analysis with kallisto

3. Analysis with kallisto#

RumBall also supports kallisto for RNA-seq analysis. The following example shows how to run kallisto on the same samples in Tutorial (human, RSEM).

3.1. Mapping reads by kallisto#

Make index for kallisto:

build=GRCh38  # specify the build that you need
Ddir=Ensembl-$build/
ncore=12  # number of CPUs
build-index-RNAseq.sh -p $ncore kallisto $build $Ddir

Run kallisto:

ID=("SRR710092" "SRR710093" "SRR710094" "SRR710095")
NAME=("HEK293_Control_rep1" "HEK293_Control_rep2" "HEK293_siCTCF_rep1" "HEK293_siCTCF_rep2")

index=$Ddir/kallisto-indexes/genome
ncore=12  # number of CPUs
mkdir -p log
for ((i=0; i<${#ID[@]}; i++))
do
    echo ${NAME[$i]}
    fq1=fastq/${ID[$i]}_1.fastq.gz
    fq2=fastq/${ID[$i]}_2.fastq.gz
    kallisto.sh -p $ncore ${NAME[$i]} "$fq1 $fq2" $Ddir reverse
done

Then you can merge the output of kallisto to make a single count matrix using kallisto_merge.sh:

s=""
for ((i=0; i<${#ID[@]}; i++))
do
    s="$s kallisto/${NAME[$i]}/abundance.tsv"
done

mkdir -p Matrix_kallisto
kallisto_merge.sh "$s" Matrix_kallisto/HEK293 $Ddir

3.2. Differential analysis#

The differential analysis step is the same with the STAR example in Tutorial (human, RSEM). Add the -k option to DESeq2.sh and edgeR.sh to use the output of kallisto_merge.sh as input.

Ctrl="kallisto/HEK293_Control_rep1 kallisto/HEK293_Control_rep2"
siCTCF="kallisto/HEK293_siCTCF_rep1 kallisto/HEK293_siCTCF_rep2"

# For DESeq2
mkdir -p Matrix_edgeR_kallisto
kallisto_merge.sh "$Ctrl $siCTCF" Matrix_deseq2_kallisto/HEK293 $Ddir
DESeq2.sh -k Matrix_deseq2_kallisto/HEK293 2:2 Control:siCTCF Human

# For edgeR
mkdir -p Matrix_deseq2_kallisto
kallisto_merge.sh "$Ctrl $siCTCF" Matrix_edgeR_kallisto/HEK293 $Ddir
edgeR.sh -k Matrix_edgeR_kallisto/HEK293 2:2 Control:siCTCF Human

Note

It is recommended to use sleuth for the differential analysis of the kallisto output instead of edgeR and DESeq2. See the sleuth walkthroughs for more details.

Contents