Processing Log M3W4

Introduction

This file serves as a processing log for the important commands used in workshop 4 for Module 3 in MB5370 in order to assemble a meta genome of BBD using a smaller subset of genomic data taken from a previous PhD project

The following steps are taken to assemble the meta genome: - Assembly w/flye - Binning w/metabat - Bin quality check w/checkm - Selecting bins based on checkm results - Taxonomic classification with gtdbtk - Finding and annotating genes with prokka - Inferring metabolic pathways

Assembly with flye

First we must assemble the meta genome of the BBD community

flye --nano-hq bbd_0.1.fastq.gz --threads 12 --out-dir bbd --meta

Binning with metabat

Next we must bin the data

metabat2 -i assembly_bbd_ordered.fasta -a depths_bbd.txt --cvExt -o bins_bbd/metabat --seed 5

Check bin quality with checkm

Next we use checkm to measure metrics such as the completeness and contamination of the bins to ensure the data is reliable

shopt -s expand_aliases
alias checkm='apptainer run -B /pvol/:/pvol /pvol/data/sif/checkm.sif checkm'

checkm lineage_wf bins_bbd out -t 4 -x fa -f checkm_results_bbd.txt

Select Bins based on checkm results with gtdbtk

Next we use awk to bring up bins with a completeness greater than 15% to look at best assembled members of BBD community Then perform taxonomic classification with gtdbtk on these bins

shopt -s expand_aliases
alias gtdbtk='apptainer run -B /pvol:/pvol,/pvol/data/mg/gtdbtkdata/release207_v2/:/refdata /pvol/data/sif/gtdbtk.sif'

gtdbtk classify_wf -x fa --cpus 2 --genome_dir genomes --out_dir out --skip_ani_screen

Find and annotate genes with prokka

Next we must identify the genes present within the genomes of bbd taxa. Prokka will identify gene sequences and identify homologous genes with known functions.

shopt -s expand_aliases
alias prokka='apptainer run -B /pvol/:/pvol /pvol/data/sif/prokka.sif prokka'

while read bin;do 
  prokka --outdir $bin --prefix $bin "../metabat/bins_bbd/${bin}.fa";
done < ../checkm/bbd_topbins.txt

Infer metabolic pathways with minpath

Next we will use minpath to take functional info for individual genes and use this to infer the presence of broader biological pathways. Will run this on each of the genomes (bins), minpath will then look at all of the annotated genes in our data and infer the minimal set of biological pathways that can explain the presence of all these genes.

while read bin;do 
  minpath -ec ${bin}.ec -report ${bin}.ec.report -details ${bin}.ec.details
done < ../checkm/bbd_topbins.txt

There are two paths of interest from the minpath output:

The first pathway is named photosynthesis light reactions (metacyc code: PWY-101). This pathway includes the essential components of photosynthesis that occur in light, Photosystem I and Photosystem II.

The second pathway is called superpathway of tetrathionate reduction (metacyc code: PWY-5360). This pathway is important because it includes enzymes required to reduce sulfite to hydrogen sulfide.

We must search for both across all bins

grep 'PWY-101' *.ec.report

grep 'PWY-5360' *.ec.report

Finally we can cross reference results with the output from gtdbtk to see the taxonomy of bins containing these pathways