MetaWRAP is a modular pipeline for shotgun metagenomic data analysis. It deploys state-of-the-art software to handle metagenomic data processing starting from raw sequencing reads and ending in metagenomic bins and their analysis. It includes hybrid algorithms that leverage the strengths of a variety of software to extract and refine high-quality bins from metagenomic data through bin consolidation and reassembly.
Allocate an interactive session and run the program. Sample session:
[user@biowulf]$ sinteractive --mem=4g --gres=lscratch:20 -c8 [user@cn0911 ~]$module load metawrap [+] Loading blast 2.14.0+ ... [+] Loading bwa 0.7.17 on cn4272 [+] Loading bowtie 2-2.3.5 [+] Loading kraken 1.1 [+] Loading kronatools 2.8.1 on cn4272 [+] Loading perl 5.34.0 on cn4272 [+] Loading samtools 1.9 ... [+] Loading singularity 4.0.1 on cn4272 [+] Loading salmon 1.7.0 [+] Loading prokka 1.14.6 [+] Loading metabat 2.15 on cn4272 [+] Loading quast 5.2.0 on cn4272 [+] Loading cutadapt 4.4 [+] Loading eigen 3.4.0-1092574b ... [+] Loading trimgalore 0.6.6 ... [+] Loading fastqc 0.11.8 [+] Loading checkm2 1.0.2 [+] Loading megahit, version 1.2.9... [+] Loading spades 3.15.5 [+] Loading gcc 11.3.0 ... [+] Loading HDF5 1.12.2 [+] Loading netcdf 4.9.0 [+] Loading openmpi/4.1.3/gcc-11.3.0 ... [+] Loading pandoc 2.18 on cn4272 [+] Loading pcre2 10.40 [+] Loading R 4.3.0 [+] Loading metawrap 1.3.2 ... [user@cn0911 ~]$mkdir /data/$USER/metawrap && cd /data/$USER/metawrap [user@cn0911 ~]$cp -r $MW_SRC/* .Download sample data, unzip the data and place them into folder RAW_READS:
[user@cn0911 ~]$cp $MW_DATA/* . [user@cn0911 ~]$gunzip *.gz [user@cn0911 ~]$mkdir RAW_READS && mv *fastq RAW_READS [user@cn0911 ~]$ls RAW_READS ERR011347_1.fastq ERR011347_2.fastq ERR011348_1.fastq ERR011348_2.fastq ERR011349_1.fastq ERR011349_2.fastqPerform the analysis steps that are described in more details in the Usage Tutorial:
[user@cn0911 ~]$mkdir READ_QC
[user@cn0911 ~]$metawrap read_qc -1 RAW_READS/ERR011347_1.fastq -2 RAW_READS/ERR011347_2.fastq -t 24 -o READ_QC/ERR011347
[user@cn0911 ~]$metawrap read_qc -1 RAW_READS/ERR011348_1.fastq -2 RAW_READS/ERR011348_2.fastq -t 24 -o READ_QC/ERR011348
[user@cn0911 ~]$metawrap read_qc -1 RAW_READS/ERR011349_1.fastq -2 RAW_READS/ERR011349_2.fastq -t 24 -o READ_QC/ERR011349
[user@cn0911 ~]$mkdir CLEAN_READS
[user@cn0911 ~]$for i in READ_QC/*; do
b=${i#*/}
mv ${i}/final_pure_reads_1.fastq CLEAN_READS/${b}_1.fastq
mv ${i}/final_pure_reads_2.fastq CLEAN_READS/${b}_2.fastq
done
Step 2: Assemble the metagenomes with the metaWRAP-Assembly module:
[user@cn0911 ~]$cat CLEAN_READS/ERR*_1.fastq > CLEAN_READS/ALL_READS_1.fastq
[user@cn0911 ~]$cat CLEAN_READS/ERR*_2.fastq > CLEAN_READS/ALL_READS_2.fastq
[user@cn0911 ~]$metawrap assembly -1 CLEAN_READS/ALL_READS_1.fastq \
-2 CLEAN_READS/ALL_READS_2.fastq \
-m 200 -t 96 --use-metaspades -o ASSEMBLY
Step 3: Run Kraken module on both reads and the assembly:
[user@cn0911 ~]$metawrap kraken -o KRAKEN -t 96 -s 1000000 CLEAN_READS/ERR*fastq ASSEMBLY/final_assembly.fastaStep 4: Bin the co-assembly with three different algorithms with the Binning module:
[user@cn0911 ~]$metawrap binning -o INITIAL_BINNING -t 96 -a ASSEMBLY/final_assembly.fasta \
--metabat2 --maxbin2 --concoct CLEAN_READS/ERR*fastq
Step 5: Consolidate bin sets with the Bin_refinement module:
[user@cn0911 ~]$metawrap bin_refinement -o BIN_REFINEMENT -t 96 \
-A INITIAL_BINNING/metabat2_bins/ \
-B INITIAL_BINNING/maxbin2_bins/ \
-C INITIAL_BINNING/concoct_bins/ \
-c 50 -x 10
Step 6: Visualize the community and the extracted bins with the Blobology module:
[user@cn0911 ~]$metawrap blobology -a ASSEMBLY/final_assembly.fasta \
-t 96 -o BLOBOLOGY \
--bins BIN_REFINEMENT/metawrap_50_10_bins CLEAN_READS/ERR*fastq
Step 7: Find the abundaces of the draft genomes (bins) across the samples:
[user@cn0911 ~]$metawrap quant_bins -b BIN_REFINEMENT/metawrap_50_10_bins \
-o QUANT_BINS \
-a ASSEMBLY/final_assembly.fasta CLEAN_READS/ERR*fastq
Step 8: Re-assemble the consolidated bin set with the Reassemble_bins module:
[user@cn0911 ~]$metawrap reassemble_bins -o BIN_REASSEMBLY \
-1 CLEAN_READS/ALL_READS_1.fastq \
-2 CLEAN_READS/ALL_READS_2.fastq \
-t 96 -m 800 -c 50 -x 10 \
-b BIN_REFINEMENT/metawrap_50_10_bins
Step 9: Determine the taxonomy of each bin with the Classify_bins module:
[user@cn0911 ~]$metawrap classify_bins -b BIN_REASSEMBLY/reassembled_bins \
-o BIN_CLASSIFICATION -t 48
Step 10: Functionally annotate bins with the Annotate_bins module
[user@cn0911 ~]$metaWRAP annotate_bins -o FUNCT_ANNOT -t 96 -b BIN_REASSEMBLY/reassembled_bins/End the interactive session:
[user@cn0911 ~]$ exit salloc.exe: Relinquishing job allocation 46116226 [user@biowulf ~]$