TCRQuant

Installation:

Installing the GitHub Repository

git clone 

Installing MiXCR

wget -c https://github.com/milaboratory/mixcr/releases/download/v3.0.13/mixcr-3.0.13.zip
unzip mixcr-3.0.13.zip
cd cd mixcr-3.0.13/
./mixcr

The path of executable file ./mixcr can be added to the .bashrc file. Detailed installation steps can be followed from the MiXCR installation manual.

Downsampling

This is a sample bash script that can be used to downsample. The users should change the directory to the working directory with fastq.gz files.

cd /presnt/working/directory
down="yes"
if [ ! $down == "no" ]
then
        #re='^[0-9]+$'
        if [ $down == "yes" ]
        then
                echo $down
                #subsample
                for i in `ls *.fastq.gz`; do echo $(zcat ${i} | wc -l)/4|bc; done > count.txt
                count=$(sort -r count.txt | tail -1)
                echo ${count}
        else
                count=$(($down + 0))
                echo ${count}
        fi
        gzip -d *.fastq.gz
        for file in $(ls *.fastq | rev | cut -c 8- | rev | uniq)
        do
                paste ${file}1.fastq ${file}2.fastq | awk '{ printf("%s",$0); n++;
                if(n%4==0) { printf("\n");} else { printf("\t");} }' |
                awk -v k=${count} 'BEGIN{srand(systime() + PROCINFO["pid"]);}{s=x++<k?x-1:int(rand()*x);if(s<k)R[s]=$0}END{for(i in R)print R[i]}' |
                awk -F"\t" '{print $1"\n"$3"\n"$5"\n"$7 > "'${file}'_sub_1.fastq"; print $2"\n"$4"\n"$6"\n"$8 > "'${file}'_sub_2.fastq"}'
                rm ${file}1.fastq ${file}2.fastq
        done
        gzip *.fastq

MiXCR

MiXCR files can be put in the same directory. The typical input files are in the fastq.gz format. A sample code to run MiXCR on your samples is her

echo "Starting Pre-processing.."

for file in $(ls *.fastq.gz | rev | cut -c 11- | rev | uniq)
do

	echo "Starting" ${file}
	echo "Species :" ${1}

	/opt/mixcr/mixcr-3.0.13 align -t 4 -s ${1} -r ${file}_align.log -OallowPartialAlignments=true -p rna-seq ${file}1.fastq.gz ${file}2.fastq.gz ${file}.vdjca > /dev/null
	cat ${file}_align.log
	/opt/mixcr/mixcr-3.0.13 assemblePartial ${file}.vdjca ${file}_contigs.vdjca > /dev/null
	/opt/mixcr/mixcr-3.0.13 assemble -t 4 ${file}_contigs.vdjca ${file}.clns > /dev/null
	/opt/mixcr/mixcr-3.0.13 exportClones ${file}.clns ${file}.txt > /dev/null

	echo ${file} "done"

done

echo "MixCR done"

More generally

mixcr align -t 4 -s <Species name> -r <Log file name> -OallowPartialAlignments=true -p rna-seq <Forward Read fastq.gz> <Reverse Read fastq.gz> <file name>.vdjca
mixcr assemblePartial <file name>.vdjca <file name>_contigs.vdjca 
mixcr assemble -t 4 <file name>_contigs.vdjca <file name>.clns 
mixcr exportClones <file name>.clns <file name>.txt

Processing files for TCRQuant

The <file name>.txt files can be grouped into two classes A and B. The files should be present in directories say Afiles and Bfiles.

TCRQuant

The final step of the analysis is to run TCRQuant. The arguements for running the program are:

1. -pA or "--path_to_fileA", "Path to input meta file list Group A (required)
2. -pB or "--path_to_fileB", "Path to input meta file list Group B (required)
3. -d or "--cwd", "Working directory path"

Rscript ranalysis.R -d . -f no -pA /path/to/Afiles -pB /path/to/Afiles