mGEMS

Bacterial sequencing data binning on strain-level based on probabilistic taxonomic classification.

More about mGEMS in the article Bacterial genomic epidemiology with mixed samples in Microbial Genomics.

Installation

In addition to mGEMS, to run the binning pipeline, you will likely need a program that pseudoalignment and another program that estimates an assignment probability matrix for the reads to the alignment targets. Please see the Dependencies subsection for more details.

Conda

Install mGEMS from bioconda with

conda install -y -c bioconda -c conda-forge -c defaults mgems

check that the installation succeeded by running

mGEMS

mGEMS binaries

Precompiled binaries are available for

Compiling from source

Requirements

C++17 compliant compiler.
cmake
git

Compilation

Clone the repository

git clone https://github.com/PROBIC/mGEMS.git

enter the directory and run

mkdir build
cd build
cmake ..
make

This will compile the mGEMS executable in the build/bin/ directory.

Dependencies

We recommend to use Themisto (v2.0.0 or newer) for pseudoalignment and mSWEEP (v1.3.2 or newer) for estimating the probability matrix.

For assembling the bins output by mGEMS, we recommend shovill for typical use-cases but metagenomic assemblers like MEGAHIT may perform better when the differences between the bins are especially small (see Supplementary Figure 2 of the mGEMS preprint). Shovill comes with an option to use different assemblers as the backend (default is SPAdes).

mSWEEP and shovill can be easily installed from bioconda.

Usage

mGEMS

The mGEMS executable provides three commands: mGEMS, mGEMS bin, and mGEMS extract. The first command (mGEMS) is shorthand for running both mGEMS bin and mGEMS extract, which bin the reads in the input pseudoalignment (mGEMS bin) and extract the binned reads from the original mixed samples (mGEMS extract).

Tutorial — E. coli ST131 sublineages

A tutorial for reproducing the E. coli ST131 sublineage phylogenetic tree presented in Mäklin et al. 2020 using mGEMS is available in the docs folder of this repository.

Quickstart — full pipeline

Index the reference sequences

Build a Themisto index to align against.

mkdir themisto_index
mkdir themisto_index/tmp
themisto build -k 31 -i example.fasta -o themisto_index/index --temp-dir themisto_index/tmp

Pseudoalign the reads

Align paired-end reads ‘reads_1.fastq.gz’ and ‘reads_2.fastq.gz’ with Themisto (note the –sort-output flag must be used!)

themisto pseudoalign -q reads_1.fastq.gz -o pseudoalignments_1.aln -i themisto_index/index --temp-dir themisto_index/tmp --rc --n-threads 16 --sort-output --gzip-output
themisto pseudoalign -q reads_2.fastq.gz -o pseudoalignments_2.aln -i themisto_index/index --temp-dir themisto_index/tmp --rc --n-threads 16 --sort-output --gzip-output

Estimate the relative abundances with mSWEEP (reference_grouping.txt should contain the groups the sequences in ‘example.fasta’ are assigned to. See the mSWEEP usage instructions for details).

mSWEEP --themisto-1 pseudoalignments_1.aln.gz --themisto-2 pseudoalignments_2.aln.gz -o mSWEEP -i reference_grouping.txt --write-probs

Bin the reads and write all bins to the ‘mGEMS-out’ folder

mkdir mGEMS-out
mGEMS -r reads_1.fastq.gz,reads_2.fastq.gz -i reference_grouping.txt --themisto-alns pseudoalignments_1.aln.gz,pseudoalignments_2.aln.gz -o mGEMS-out --probs mSWEEP_probs.csv -a mSWEEP_abundances.txt --index themisto_index

This will write the binned paired-end reads for all groups in the mSWEEP_abundances.txt file in the mGEMS-out folder (compressed with zlib).

Advanced use

You can also extract the read-to-group assignments table that mGEMS uses internally by adding the --write-assignment-table toggle to the call to mGEMS or mGEMS bin:

mGEMS --groups group-3,group-4 -r reads_1.fastq.gz,reads_2.fastq.gz -i reference_grouping.txt --themisto-alns pseudoalignments_1.aln.gz,pseudoalignments_2.aln.gz -o mGEMS-out --probs mSWEEP_probs.csv -a mSWEEP_abundances.txt --index themisto_index --write-assignment-table

… or bin and write only the reads that are assigned to “group-3” or “group-4” by adding the ‘–groups group-3,group-4’ flag

mGEMS --groups group-3,group-4 -r reads_1.fastq.gz,reads_2.fastq.gz -i reference_grouping.txt --themisto-alns pseudoalignments_1.aln.gz,pseudoalignments_2.aln.gz -o mGEMS-out --probs mSWEEP_probs.csv -a mSWEEP_abundances.txt --index themisto_index

… write the reads that pseudoaligned to a reference sequence but were not assigned to any group by adding the --write-unassigned flag:

mGEMS --groups group-3,group-4 -r reads_1.fastq.gz,reads_2.fastq.gz -i reference_grouping.txt --themisto-alns pseudoalignments_1.aln.gz,pseudoalignments_2.aln.gz -o mGEMS-out --probs mSWEEP_probs.csv -a mSWEEP_abundances.txt --index themisto_index --write-unassigned

Alternatively, find and write only the read bins for “group-3”, “group-4”, and the reads that pseudoaligned but were not assigned to any group; skipping extracting the reads

mGEMS bin --groups group-3,group-4 --themisto-alns pseudoalignments_1.aln.gz,pseudoalignments_2.aln.gz -i reference_grouping.txt -o mGEMS-out --probs mSWEEP_probs.csv -a mSWEEP_abundances.txt --index themisto_index --write-unassigned

… and extract the reads when feeling like it

mGEMS extract --bins mGEMS-out/group-3.bin,mGEMS-out/group-4.bin,mGEMS-out/unassigned_reads.bin -r
reads_1.fastq.gz,reads_2.fastq.gz -o mGEMS-out

Accepted input flags

mGEMS accepts the following input flags

    -r                       Comma-separated list of input read(s).
    -i                       Group identifiers file used with the mSWEEP call.
    --themisto-alns          Comma-separated list of pseudoalignment file(s) 
                             for the reads from themisto.
    -o                       Output directory (must exist before running!).
    --probs                  Comma-separated Posterior probability matrix (output from mSWEEP with
                             the --write-probs flag).
    -a                       Relative abundance estimates from mSWEEP (tab-separated, 1st
                             column has the group names and 2nd column the estimates).
    --index                  Themisto pseudoalignment index directory.
    --groups                 (Optional) Which groups to extract from the input reads.
    --min-abundance          (Optional) Extract only groups that have a relative abundance higher than this value.
    --compress               (Optional) Toggle compressing the output files (default: compress)
    --write-unassigned       (Optional) Extract reads that pseudoaligned to a reference sequence but were not assigned to any group. (default: off)
    --write-assignment-table (Optional) Write the read to group assignments table to `reads_to_groups.tsv` in the output directory. (default: off).
    --unique-only            (Optional) Write only the reads that are assigned to a single group.

Citation

If you use mGEMS, please cite us as “Mäklin T, Kallonen T, Alanko J et al. Bacterial genomic epidemiology with mixed samples. Microb Genom 2021, 7:11 (https://doi.org/10.1099/mgen.0.000691)"

You should also cite the method that you used to estimate the input probability matrix to mGEMS, which is likely to be mSWEEP.

To cite a specific version of mGEMS, visit the releases page and find the doi for the version of the program that you used. Then, cite the version (v1.1.0 in the example) as “Tommi Mäklin. (2021). PROBIC/mGEMS: mGEMS-v1.1.0 (20 October 2021) (v1.1.0). Zenodo. (https://doi.org/10.5281/zenodo.5583245)". Citing the source code properly helps ensure that your analyses are reproducible. Please also cite the article if you use mGEMS.

License

The source code from this project is subject to the terms of the MIT license. A copy of the MIT license is supplied with the project, or can be obtained at https://opensource.org/licenses/MIT.