mcdr-mtb-standalone is a pipeline for prediction of drug resistance class (MDR, XDR or Susceptible) of Mycobacterium tuberculosis isolates from whole genome sequencing (WGS) data. The users can start with paired-end FASTQ files or variant calling output VCF files.
There are 4 different scripts for prediction:
- mcdr-WGS-predict.sh: Prediction of drug resistance class of single or multiple MTB WGS data (paired-end fastq files).
- mcdr-VCF-predict.sh: Prediction of drug resistance class of single MTB VCF file.
- mcdr-multi-VCF-predict.sh: Prediction of drug resistance class of multiple MTB VCF files.
- mcdr-merge-VCF-predict.sh: Prediction of drug resistance class of MTB merged VCF file.
- trim-galore (version
0.6.7) - quality check and trimming of read sequences - bwa (
0.7.17-r1188) - reference based alignment - samtools (
1.13)- processing the BAM files - freebayes (
v1.3.6) - variant calling - libvcflib-tools (
1.0.7) - processing the VCF files - libvcflib-dev (
1.0.7) - processing the VCF files - bgzip (
1.13+ds) - zipping files - R (
4.2.2 Patched (2022-11-10 r83330)) - data operation and machine learning model usage
(For ubuntu)
sudo apt-get install trim-galore bwa samtools freebayes libvcflib-tools libvcflib-dev bgzip
(For other distributions)
The installation steps for the different packages/tools are given in the following links:
- trim-galore - https://github.com/FelixKrueger/TrimGalore
- bwa - https://github.com/lh3/bwa
- samtools, bcftools, bgzip(htstools) - http://www.htslib.org/download/
- freebayes - https://github.com/freebayes/freebayes
- vcflib - https://github.com/vcflib/vcflib
R should be installed in the user system/PC. R installation steps are given in https://cran.r-project.org/.
Create a clone of the repository:
git clone https://github.com/AbhirupaGhosh/mcdr-mtb-standalone
Note: Creating a clone of the repository requires git to be installed.
The git can be installed using
sudo apt-get install git
OR
Download using wget:
wget https://github.com/AbhirupaGhosh/mcdr-mtb-standalone/archive/refs/heads/main.zip
unzip main.zip
Note: wget can be installed using
sudo apt-get install wget
chmod +x INSTALLATION_DIR/mcdr-mtb-standalone config.sh mcdr-WGS-predict.sh mcdr-VCF-predict.sh mcdr-multi-VCF-predict.sh mcdr-merge-VCF-predict.sh
INSTALLATION_DIR = Directory where mcdr-mtb-standalone is installed
The config.sh looks like
freebayes_path=/usr/bin/freebayes
samtools_path=/usr/bin/samtools
bwa_path=/usr/bin/bwa
trim_galore_path=/usr/bin/trim_galore
vcflib_path=/usr/bin/vcflib
bgzip_path=/usr/bin/bgzip
bcftools_path=/usr/bin/bcftools
trim_galore_cores=4
bwa_mem_cores=4
samtools_cores=4
Note: It shows the default paths of the executables files for freebayes, samtools, bwa, trim galore!, vcflib, bgzip and bcftools. The users need to update the paths of the executables, in case these tools were installed in ways other than the apt-get install command.
Initially change the directory to the directory where mcdr-mtb-standalone is installed.
cd INSTALLATION_DIR/mcdr-mtb-standalone
Different operations can be performed by calling the appropriate scripts with two command-line arguments: INPUT_DIR and OUTPUT_DIR.
INPUT_DIR = the path (absolute or relative) of the folder containing the input files.
OUTPUT_DIR = the path (absolute or relative) of the folder in which mcdr-mtb-standalone will store the outputs.
The executable script, and contents of INPUT_DIR and OUTPUT_DIR depends on the choice of operations. The different operations are explained below.
(.fastq to drug resistance class (M, S, X))
./mcdr-WGS-predict.sh INPUT_DIR OUTPUT_DIR
INPUT_DIR must contain paired end FASTQ (ISOLATE1_1.fastq.gz & ISOLATE1_2.fastq.gz) of 1 or more isolates.
OUTPUT_DIR will contain
- one folder for each ISOLATE ID (ISOLATE_DIR). Each folder will contain
- the VCF file (ISOLATE1.vcf)
- the intermediate BAM files (ISOLATE1.bam, ISOLATE1_sorted.bam)
- the MERGED.vcf file (only in case of multiple isolates)
- the intermediate TSV file (ISOLATE1.tsv/MERGED.tsv)
Note: The merged.vcf file will not be present if there was only one isolate in the INPUT_DIR.
The prediction output will be displayed on the terminal.
(.vcf to drug resistance class (S, M, X))
./mcdr-VCF-predict.sh INPUT_DIR OUTPUT_DIR
INPUT_DIR must contain VCF file (ISOLATE1.vcf)
OUTPUT_DIR will contain an intermediate .tsv file (ISOLATE1.tsv)
The prediction output will be displayed on the terminal.
(multiple .vcf to drug resistance class (S, M, X))
./mcdr-multi-VCF-predict.sh INPUT_DIR OUTPUT_DIR
INPUT_DIR must contain more than 1 VCF file (ISOLATE1.vcf, ISOLATE2.vcf…. ISOLATEn.vcf)
OUTPUT_DIR will contain
- the MERGED.vcf file
- the intermediate .tsv file (MERGED.tsv)
The prediction output will be displayed on the terminal.
(merged.vcf to drug resistance class (S, M, X))
./mcdr-merge-VCF-predict.sh INPUT_DIR OUTPUT_DIR
INPUT_DIR must contain a merged VCF file (MERGED.vcf) with information of one or more isolates.
OUTPUT_DIR will contain the intermediate .tsv file (MERGED.tsv)
The prediction output will be displayed on the terminal.
Step 1. Create an Input directory
mkdir /home/username/Input_Dir1
Step 2. Get Data
Download the whole genome sequencing FASTQ files of a MTB isolate run, ERR137249 (ERR137249_1.fastq & ERR137249_2.fastq) from https://www.ebi.ac.uk/ena/browser/view/ERR137249
Step 3. Store these files in Input_Dir1
Step 4. Create an Output directory
mkdir /home/username/Output_Dir1
Step 5. Go to the mcdr-mtb-standalone installation directory
cd /home/username/Documents/mcdr-mtb-standalone/
Step 6. Run mcdr-WGS-predict.sh
./mcdr-WGS-predict.sh /home/username/Input_Dir1/ /home/username/Output_Dir1/
Input_Dir1 contains ERR137249_1.fastq, ERR137249_2.fastq
Output_Dir1 contains -
- Folder - ERR137249
- ERR137249.tsv
The ERR137249 folder contains -
- reference folder - reference genome and index files
- Trim galore outputs - ERR137249_1_val_1.fq.gz, ERR137249_2_val_2.fq.gz, ERR137249_1_trimming_report.txt, ERR137249_2_trimming_report.txt
- Bwa-mem output - ERR137249.bam
- Intermediate BAM files - ERR137249_fix.bam, ERR137249_namesort.bam, ERR137249_positionsort.bam, ERR137249_markdup.bam
- BAM index - ERR137249.bam.bai
- Freebayes output - ERR137249.vcf
- VCF compressed - ERR137249.vcf.gz
- VCF index - ERR137249.vcf.gz.csi
Printed Output -
ISOLATE PREDICTION DIFF RI
1 ERR137249 S 0.04866350 0
Step 1. Create an Input directory
mkdir /home/username/Input_Dir2
Step 2. Get Data
Download the whole genome sequencing FASTQ files of MTB ISOLATE runs, ERR137249 (ERR137249_1.fastq & ERR137249_2.fastq) and SRR1103491 (SRR1103491_1.fastq & SRR1103491_2.fastq) from https://www.ebi.ac.uk/ena/browser/view/ERR137249 and https://www.ebi.ac.uk/ena/browser/view/SRR1103491
Step 3. Store these files in Input_Dir2
Step 4. Create an Output directory
mkdir /home/username/Output_Dir2
Step 5. Go to the mcdr-mtb-standalone installation directory
cd /home/username/Documents/mcdr-mtb-standalone/
Step 6. Run mcdr-WGS-predict.sh
./mcdr-WGS-predict.sh /home/username/Input_Dir1/ /home/username/Output_Dir2/
Input_Dir2 contains ERR137249_1.fastq, ERR137249_2.fastq, SRR1103491_1.fastq, SRR1103491_2.fastq
Output_Dir2 contains -
- Two folders - ERR137249, SRR1103491
- merged.vcf
- merged.tsv
Each of the ERR137249 and SRR1103491 named folder contains -
- reference folder - reference genome and index files
- Trim galore outputs - ISOLATENAME_1_val_1.fq.gz, ISOLATENAME_2_val_2.fq.gz, ISOLATENAME_1_trimming_report.txt, ISOLATENAME_2_trimming_report.txt
- Bwa-mem output - ISOLATENAME.bam
- Intermediate BAM files - ISOLATENAME_fix.bam, ISOLATENAME_namesort.bam, ISOLATENAME_positionsort.bam, ISOLATENAME_markdup.bam
- BAM index - ISOLATENAME.bam.bai
- Freebayes output - ISOLATENAME.vcf
- VCF compressed - ISOLATENAME.vcf.gz
- VCF index - ISOLATENAME.vcf.gz.csi
Printed Output -
ISOLATE PREDICTION DIFF RI
1 ERR137249 S 0.04866350 0
2 SRR1103491 M 0.02846038 0
Step 1. Create an Input directory
mkdir /home/username/Input_Dir3
Step 2. Store a VCF file generated from variant calling of a MTB isolate based on MTB H37Rv reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/195/955/GCF_000195955.2_ASM19595v2/GCF_000195955.2_ASM19595v2_genomic.fna.gz)
Step 3. Create an Output directory
mkdir /home/username/Output_Dir3
Step 4. Go to the mcdr-mtb-standalone installation directory
cd /home/username/Documents/mcdr-mtb-standalone/
Step 5. Run mcdr-VCF-predict.sh
./mcdr-VCF-predict.sh /home/username/Input_Dir3/ /home/username/Output_Dir3/
Input_Dir3 contains ERR137249.vcf
Output_Dir3 contains - ERR137249.tsv
Printed Output -
ISOLATE PREDICTION DIFF RI
1 ERR137249 S 0.04866350 0
Step 1. Create an Input directory
mkdir /home/username/Input_Dir4
Step 2. Store multiple VCF files generated from variant calling of MTB isolates based on MTB H37Rv reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/195/955/GCF_000195955.2_ASM19595v2/GCF_000195955.2_ASM19595v2_genomic.fna.gz)
Step 3. Create an Output directory
mkdir /home/username/Output_Dir4
Step 4. Go to the mcdr-mtb-standalone installation directory
cd /home/username/Documents/mcdr-mtb-standalone/
Step 5. Run mcdr-multi-VCF-predict.sh
./mcdr-multi-VCF-predict.sh /home/username/Input_Dir4/ /home/username/Output_Dir4/
Input_Dir4 contains ERR137249.vcf, SRR1103491.vcf
Output_Dir4 contains -
- Compressed VCFs - ERR137249.vcf.gz and SRR1103491.vcf.gz
- VCF indices - ERR137249.vcf.gz.csi and SRR1103491.vcf.gz.csi
- Merged.vcf
- Merged.tsv
Printed Output -
ISOLATE PREDICTION DIFF RI
1 ERR137249 S 0.04866350 0
2 SRR1103491 M 0.02846038 0
Step 1. Create an Input directory
mkdir /home/username/Input_Dir5
Step 2. Store a merged VCF file generated by merging multiple VCF files from variant calling of MTB isolates based on MTB H37Rv reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/195/955/GCF_000195955.2_ASM19595v2/GCF_000195955.2_ASM19595v2_genomic.fna.gz)
Step 3. Create an Output directory
mkdir /home/username/Output_Dir5
Step 4. Go to the mcdr-mtb-standalone installation directory
cd /home/username/Documents/mcdr-mtb-standalone/
Step 5. Run mcdr-merge-predict.sh
./mcdr-merge-predict.sh /home/username/Input_Dir5/ /home/username/Output_Dir5/
Input_Dir5 contains merged.vcf
Output_Dir5 contains Merged.tsv
Printed Output -
ISOLATE PREDICTION DIFF RI
1 ERR137249 S 0.04866350 0
2 SRR1103491 M 0.02846038 0
Abhirupa Ghosh, Sudipto Bhattacharjee and Sudipto Saha
The scripts were tried and tested on the Ubuntu Operating system.
This tool is strictly for Research Use Only. By using this tool the user acknowledges no intended medical purpose such as patient diagnosis.