Metagenome binning using MetaBAT

Authored by Fan Yang Adapted by Adelaide Rhodes, Ph.D. for Metagenomics I & II

EDAMAME-2016 wiki


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Overarching Goal

  • This tutorial will contribute towards an understanding of microbial metagenome analysis

Learning Objectives

  • Understand metagenomic binning and perform a binning exercise

Tutorial

We will be running a specific tool for metagenomic binning called MetaBAT. You can read about this tool and its applications in the original paper, [MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities]

This tutorial takes place assuming you have an assembled metagenome and its mapped read abundances.

1. Preparing to bin

So you learned about how to assemble sequences yesterday and assembled some contigs, for today’s tutorial, we need a full assembled file. Let’s also get some assembled and mapped data.

cd ~
mkdir data && cd ~/data
wget https://s3.amazonaws.com/edamame/SRR492066.sam.bam.sorted.bam
wget https://s3.amazonaws.com/edamame/SRR492065.sam.bam.sorted.bam
wget https://s3.amazonaws.com/edamame/final.contigs.fa``
Q: Can you think of a reason why we couldn’t use the partial
assembled contigs from the metagenome assembly exercise?

2. Now let’s do some binning!

cd ~/data

nano binscript.sh

Copy and paste the following block into the script

#!/bin/bash

for i in *.sorted.bam
do
    mkdir ~/data/$i.bins
    cd ~/data/$i.bins
    ~/metabat/bin/runMetaBat.sh ~/data/final.contigs.fa ~/data/$i
done

Control-X to exit

chmod 775 binscript.sh

binscript.sh

3. What if we want to bin the contigs with different threshold?

cd ~/data/SRR492065.sam.bam.sorted.bam.bins
#First, try sensitive mode to better sensitivity
~/metabat/bin/metabat -i ~/data/final.contigs.fa -a final.contigs.fa.depth.txt -o bin1 --sensitive -l -v --saveTNF saved.tnf --saveDistance saved.gprob

#Try specific mode to improve specificity further; this time the binning will be much faster since it reuses saved calculations
~/metabat/bin/metabat -i ~/data/final.contigs.fa -a final.contigs.fa.depth.txt -o bin2 --specific -l -v --saveTNF saved.tnf --saveDistance saved.gprob

#Try specific mode with paired data to improve sensitivity while minimizing the loss of specificity
~/metabat/bin/metabat -i ~/data/final.contigs.fa -p final.contigs.fa.paired.txt -o bin3 --specific -l -v --saveTNF saved.tnf --saveDistance saved.gprob

4. Simple annotation strategies - kraken

If you have a simple data set with very common bacteria, you can jump right into kraken for annotation here.

cd ~/data/cd SRR492065.sam.bam.sorted.bam.bins/

/local/cluster/kraken/kraken-0.10.5-beta/kraken --db /local/cluster/kraken/krakenfullDB_20151215/KRAKEN/ --threads8 --fasta-input final.contigs.fa.metabat-bins-.1.fa --output bin1.kraken


/local/cluster/kraken/kraken-0.10.5-beta/kraken-translate --db /local/cluster/kraken/krakenfullDB_20151215/KRAKEN/ bin1.kraken > bin1.kraken.labels

kraken has now provided a taxonomic assignment to all of the clusters.

“Kraken is a system for assigning taxonomic labels to short DNA sequences, usually obtained through metagenomic studies. Kraken aims to achieve high sensitivity and high speed by utilizing exact alignments of k-mers and a novel classification algorithm.

For a simulated metagenome of 100 bp reads in its fastest mode of operation, , Kraken processed over 4 million reads per minute on a single core, over 900 times faster than Megablast and over 11 times faster than the abundance estimation program MetaPhlAn. Kraken’s accuracy is comparable with Megablast, with slightly lower sensitivity and very high precision.” Citation

See Kraken Home Page for more information.

5. Functional annotation strategies - prodigal

Prodigal (Prokaryotic Dynamic Programming Genefinding Algorithm) is a microbial (bacterial and archaeal) gene finding program developed at Oak Ridge National Laboratory and the University of Tennessee. See the Prodigal home page for more info. Citation

Using prodigal with the same set of data, we can get a list of predicted genes.

prodigal -p meta -a final.contigs.genes.bin1.faa -d final.contigs.genes.bin1.fna -f gff -o final.contigs.genes.bin1.gff -i final.contigs.fa.metabat-bins-.1.fa

Kevin will talk more next week about constructing gene orthologies using Hmm search strategies.