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Binnacle - estimates coverage for graph scaffolds and integrates with existing metagenome binning tools
Binnacle accurately computes coverage of graph scaffolds and seamlessly integrates with leading binning methods such as MetaBAT2, MaxBin 2.0, and CONCOCT. Using graph scaffolds, as opposed to contigs (most common approach) for binning improves the contiguity and quality of metagenomic bins and can capture a broader set of the accessory elements of the reconstructed genomes.
To run Binnacle, you will need Python 3.7.x, Bedtools, Samtools, BioPython, networkx, numpy, and Pandas.
An Environment.yml file is available and this can be used to create a conda environment that is suitable to run binnacle.
The detailed documentation about how to install these packages is given here.
We use graph scaffolds that are output of MetaCarvel scaffolding tool, so you will also need to download and install MetaCarvel. There is a step by step installation guide for MetaCarvel.
Generally, when you have one or multiple metagenomic samples, we need to assemble, scaffold, and bin contigs/scaffolds from each sample to generate metagenomic bins. We recommend using Megahit for assembly, and MetaCarvel for scaffolding. We provide a helper guide to work through assembly, scaffolding, and per-base coverage estimation steps here.
Follow these steps to generate files for running binning methods with graph scaffolds:
- Generate accurate scaffolds (mis-scaffolding module), and estimate scaffold span and coverage.
This step takes graph scaffolds from MetaCarvel output directory, and coverage estimated for contigs using reads from the same sample as input. It outputs coverage estimates for accurate set of graph scaffolds along with other necessary information about its coordinates, orientation, etc in the output directory. Contig level coverages can be presented to binnacle either as a text file describing the perbase coverage or bam and bed files of the read alignments.
python Estimate_Abundances.py -g [ORIENTED.gml] -a [COVERAGE_SORTED.txt] -c [CONTIGS.fa] -d [OUTPUT_DIRECTORY]
usage: Estimate_Abundances.py [-h] [-g ASSEMBLY] [-a COVERAGE] [-bam BAMFILE]
[-bed BEDFILE] [-c CONTIGS] -d DIR [-o COORDS]
[-w WINDOW_SIZE] [-t THRESHOLD]
[-n NEIGHBOR_CUTOFF] [-p POSCUTOFF]
[-pre PREFIX]
binnacle: A tool for binning metagenomic datasets using assembly graphs and
scaffolds generated by metacarvel. Estimate_Abundances.py estimates abundance
for scaffolds generated by MetaCarvel. If the coordinates computed by binnacle
is specified then the abundance for each scaffold is estimated based on the
contig abundances and the coordinates. If the coordinates are not specified
then binnacle etimates the abundance from scratch. While calculating all vs
all abundances please specify the coordinates(Coordinates_After_Delinking.txt)
through the "coords" parameter. The abundances can be provided as a bed file,
bam file or a text file describing the per base coverage obtained by running
the genomeCoverageBed program of the bedtools suite.
optional arguments:
-h, --help show this help message and exit
-g ASSEMBLY, --assembly ASSEMBLY
Assembly Graph generated by Metacarvel
-a COVERAGE, --coverage COVERAGE
Output generated by running genomecov -d on the bed
file generated by MetaCarvel.
-bam BAMFILE, --bamfile BAMFILE
Bam file from aligning reads to contigs
-bed BEDFILE, --bedfile BEDFILE
Bed file from aligning reads to contigs. If bed file
is provided please provide a fasta file of the contigs
-c CONTIGS, --contigs CONTIGS
Contigs generated by the assembler, contigs.fasta
-d DIR, --dir DIR output directory for results
-o COORDS, --coords COORDS
Coordinate file generated by Binnacle
-w WINDOW_SIZE, --window_size WINDOW_SIZE
Size of the sliding window for computing test
statistic to identify changepoints in coverages
(Default=1500)
-t THRESHOLD, --threshold THRESHOLD
Threshold to identify outliers (Default=99)
-n NEIGHBOR_CUTOFF, --neighbor_cutoff NEIGHBOR_CUTOFF
Filter size to identify outliers within (Defualt=100)
-p POSCUTOFF, --poscutoff POSCUTOFF
Position cutoff to consider delinking (Default=100)
-pre PREFIX, --prefix PREFIX
Prefix to be attached to all outputs
- If you want to estimate coverage of graph scaffolds of a sample from its reads you will run Estimate_Abundances.py with the following parameters,
-g Path to oriented.gml from running metacarvel on sample
-c Path to contigs obtained by assembling reads of sample
-a Coverage of contigs in ths sample by mapping to its reads -- See Wiki for how to calculate coverage information
-d Output directory
- When working with multiple samples, we use reads from the same sample to "correct" graph scaffolds and estimate its span. But, we can use reads from all other samples to estimate coverage of graph scaffolds across samples. Using information from multiple samples can help reduce noise in the binnning phase, and we highly recommennt it. So, if you want to estimate coverage of graph scaffolds (Sample 1) from the reads of another sample (Sample 2), you will run Estimate_Abundances.py with these modifications.
-a Coverage of contigs in Sample 1 by mapping reads of Sample 2 -- See Wiki for how to calculate coverage information
-o Coordinates of scaffolds from Sample 1 that you would have generated from the previous step.
-d Same output directory as Sample 1
- Once you have coverage estimated for graph scaffolds from all samples (all vs. all), we need to combine this information and generate files for running metagenome binning methods. We provide files that can be easily used with MetaBAT2, CONCOCT, and MaxBin2.0.
To generate the feature matrix for clustering, we will use Collate.py program from Binnacle. It takes the path to the output directory where all the summary information generated by the previous steps are placed and the binning method that you would like to run next.
python Collate.py -h
usage: Collate.py [-h] -d DIR [-m METHOD] [-k KEEP]
binnacle: A tool for binning metagenomic datasets using assembly graphs and
scaffolds generated by metacarvel.Estimate_Abundances.py estimates abundance
for scaffolds generated by MetaCarvel. The program Collate.py collects the
summary files generated by Estimate_Abundances.py
optional arguments:
-h, --help show this help message and exit
-d DIR, --dir DIR Output directory that contains the summary files
generated by running Estimate_Abundances.py
-m METHOD, --method METHOD
Binning method to format the output to. Presently we
support 1. Metabat 2. Maxbin 3. Concoct 4. Binnacle
(Default)
-k KEEP, --keep KEEP Retain the summary files generated by
Estimate_Abundances.py. Defaults to True
- Using the abundances.txt file based on the method selected (-m) in the previous step, you can run that binning method to generate bins for your graph scaffolds.
Please checkout the wiki for a detailed description on setting up the python environment, methods to calculate coverage and a typical workflow to run binnacle.
To visualize the graph scaffolds we recommend using MetagenomeScope which is a web-based browser. The input to metagenomescope is Assembly_Graph_Filtered.gml. Detailed documentation on installing and running MetagenomeScope is given here.
Please cite Muralidharan HS, Shah N, Meisel JS and Pop M (2021) Binnacle: Using Scaffolds to Improve the Contiguity and Quality of Metagenomic Bins. Front. Microbiol. 12:638561. doi: 10.3389/fmicb.2021.638561.
This tool is still under development. Please open issue here on Github or contact us if you have any questions.
Harihara Muralidharan: [email protected]
Nidhi Shah: [email protected]