metaWRAP for Cholera

Prefetch SRA Files

bash

# Download SRA files based on SRR numbers

prefetch SRRXXXXX

Move .sra Files to a Single Directory

bash

find . -type f -name '*.sra' -exec mv {} /data/chengz/Cohort/CFS-1/CFS-1-fastq \;

Check Folder Size

bash

du -sh *

Convert .sra Files to FASTQ Format

bash

fasterq-dump -p -e 80 --split-3 *.sra

Quality Control with MetaWRAP

bash

source activate metawrap

metawrap read_qc --skip-bmtagger -1 FLJ2_1.fq -2 FLJ2_2.fq -t 50 -o READ_QC/FLJ2

--skip-bmtagger: Skips host contamination removal.

Input files (-1 and -2) should be named as sample_1 and sample_2.

-t 50: Use 50 threads.

Merge FASTQ Files

bash

mkdir ALL

cat *_R1.fastq > ALL/ALL_READS_R1.fastq

cat *_R2.fastq > ALL/ALL_READS_R2.fastq

Remove Host Contamination

Paired-End Sequencing

bash

for i in SRR26886987 SRR26886988 ...; do

    bowtie2 -p 30 -x /mnt/database/humanindex/humangenome \

        -1 ${i}_1.fastq.gz -2 ${i}_2.fastq.gz \

        -S ${i}.sam --un-conc-gz /data/chengz/Cohort1/BangladeshControl/Fastq/${i}.fastq.gz;

done

Single-End Sequencing

bash

for i in SRR26267438 SRR26267439 ...; do

    bowtie2 -p 30 -x /mnt/database/humanindex/humangenome \

        -U ${i}.fastq.gz -S ${i}.sam \

        --un-conc-gz /data/chengz/Cohort1/IndianControl/Fastq/Output/${i}.fastq.gz;

done

Assembly with MetaWRAP

Single-End Assembly

bash

megahit -r SRR15408397_1.fastq --min-contig-len 800 -t 96 -o ASSEMBLY

Paired-End Assembly

bash

for i in *.fastq.1.gz; do

    f=$(basename "$i" .fastq.1.gz)

    metawrap assembly -1 $f.fastq.1.gz -2 $f.fastq.2.gz -t 50 -l 800 -o ASSEMBLY/${f};

done

Binning with MetaWRAP

Prepare Filenames

bash

for file in input_dir/*; do

    if [[ $file == *.fastq.1.gz ]]; then

        mv $file $(echo $file | sed 's/\.fastq\.1\.gz/_1.fastq.gz/')

    fi

done

for file in input_dir/*; do

    if [[ $file == *.fastq.2.gz ]]; then

        mv $file $(echo $file | sed 's/\.fastq\.2\.gz/_2.fastq.gz/')

    fi

done

Binning

bash

for sample in "${samples[@]}"; do

    metawrap binning -o "${sample}_BINNING" -t 40 \

        -a ASSEMBLY/${sample}/final_assembly.fasta \

        --metabat2 --maxbin2 --concoct \

        ${sample}_1.fastq ${sample}_2.fastq

done

Bin Refinement

bash

for dir in SRR*_BINNING; do

    metawrap bin_refinement -o "${dir}_REBIN" -t 30 \

        -A "${dir}/metabat2_bins/" -B "${dir}/maxbin2_bins/" -C "${dir}/concoct_bins/" \

        -c 50 -x 10;

done

Extract and Rename Refined Bins

bash

find SRR*/metawrap_50_10_bins/ -type f -name "*.fa" -exec cp {} /path/to/Output/ \;

Remove Redundant Bins with dRep

Taxonomic Classification with GTDB-Tk

bash

source activate gtdbtk

nohup gtdbtk classify_wf --genome_dir /data/chengz/Cohort1/TotalBin/BinALL --skip_ani_screen --out_dir /data/chengz/Cohort1/TotalBin/gtdbtkALL --extension fa --cpus 16 &