Bioinformatics analysis used to determine reinfection and relapse tuberculosis using whole genome sequencing data.

Citation: Utilizing whole genome sequencing to delineate relapse and reinfection tuberculosis on the Canadian prairies (https://doi.org/10.1080/24745332.2024.2366310)

Step 1: Quality check of raw FASTQ files

perl WGS_SIBP_P1.pl Prepare_Input.txt RAW_READS QualityCheck RESULTS

Step 2: Remove adapter sequences from FASTQ files

perl AdapterRem.pl Prepare_Adapter-Input.txt RAW_READS adapters.fa AdapterRemoval AdapterRemoval-QC

RAW_READS = Directory with raw FASTQ files
adapters.fa = Adapter sequences in fasta format
AdapterRemoval = Directory to save FASTQ files after adapter cleaning
AdapterRemoval-QC = Quality check results of FASTQ files after adapter cleaning

Step 3: Read mapping to the Mycobacterium tuberculosis reference genome

perl ReadMapping.pl Prepare_Input.txt GCF_000277735.2_ASM27773v2_genomic.fna RAW_READS TB_Results

RAW_READS = Directory with raw/cleaned FASTQ files
TB_Results = Directory to store alignment results

Step 4: De novo genome assembly

perl WGS_SIBP_P2.pl Prepare_Input.txt RAW_READS raw 35 Assembly

RAW_READS = Directory with raw/cleaned FASTQ files
Assembly = Directory to store assembled genomes

Step 5: Check the quality of the assembled genomes

quast.py AssembledContigs/* -r GCF_000195955.2_ASM19595v2_genomic.fna -g GCF_000195955.2_ASM19595v2_genomic.gff

AssembledContigs = Directory containing all assembled genomes in fasta format

Step 6: Coverage calculation

Program version: fastqinfo-2.0

Insert size calculation Example:

   head -n 2 C09MB033185_i7-43_R1.fastq |tail -n 2|wc -c

Coverage calculation Example:

fastq-info/bin/fastqinfo-2.0.sh -r 215 C09MB033185_i7-43_R1.fastq C09MB033185_i7-43_R2.fastq Genome.fasta
fastq-info/bin/fastqinfo-2.0.sh -r 215 C09MB033185_i7-43_R1.fastq C09MB033185_i7-43_R2.fastq ALL-Contigs/C09MB033185.fasta

Step 7: Spolingotyping

 python Spolingotyping.py

Step 8: Lineages and AMR prediction

 perl mykrobe_AMR.pl Prepare_Input.txt RAW_READS Lineage_AMR

Step 9: Single nucleotide polymorphisms (SNPs) and phylogenetic analysis

  MTBseq --step TBfull --threads 30
  raxmlHPC -s TB_joint_cf4_cr4_fr75_ph4_samples50_amended_u95_phylo_w12.plainIDs.phy -m GTRGAMMA -p 12345 -# 200 -T 35 -n RecurrentTb