mcdr-mtb-standalone

Introduction

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.

Workflows

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.

Pre-requisites

• 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

Installation

Step 1: Install dependent packages/tools

(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/.

Step 2: Install mcdr-mtb-standalone

I. Download the software from GitHub repository

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

II. Make the shell scripts executable

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

III. update the paths in config.sh (optional)

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.

Usage

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.

1) Prediction of drug resistance class of one or many MTB paired-end FASTQ files.

(.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.

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2) Prediction of drug resistance class of a single MTB VCF file.

(.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.

---

3) Prediction of drug resistance class of multiple MTB VCF files.

(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.

---

4) Prediction of drug resistance class of merged MTB VCF file.

(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.

---

Demo runs

1. Prediction from FASTQ

a. Single isolate

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

b. Multiple isolates

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

2. Prediction from single VCF

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

3. Prediction from multiple VCFs

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

4. Prediction from merged VCFs

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

Team

Abhirupa Ghosh, Sudipto Bhattacharjee and Sudipto Saha

Disclaimer

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.