Skip to content

Modular snakemake workflow for assembly-based metagenomics

License

Notifications You must be signed in to change notification settings

tanaes/snakemake_assemble

Repository files navigation

Snakemake pipeline for shotgun data QC

A Snakemake workflow for assessing the quality of many shotgun sequence samples simultaneously using Snakemake and MultiQC.

It modifies some code from jlanga's snakemake skeleton workflow, and builds on previous code I wrote in snakemake_shotqual.

Installation

Currently, this tool inherits most of its dependencies from a single Conda environment that must be loaded to execute the launch.sh script.

To create this environment, you can run the install.sh script in ./bin/install:

bash ./bin/install/install.sh

This will create a conda environment named snakemake_assemble with a number of dependencies, including the following tools:

In addition, it will download Quast and install in the snakemake_assemble environment using pip.

Note that I've had some troubles with the conda recipes for some of these packages, especially MaxBin2. There seem to be some issues with the particular Perl version specified in one of the MaxBin2 dependencies. Currently, I've been getting around this by manually reinstalling the MaxBin2 module.

Future versions of this pipeline shoud employ Snakemake's built-in per-rule package management so that the environment creation is more modular.

Usage

This workflow is meant to be modular and configurable for shotgun analyses.

In its simplest iteration, the complete set of rules can be launched for a dataset by activating the snakemake_assemble Conda environment and running the following command:

bash launch.sh ./

Behind the scenes of launch.sh, this invokes the snakemake command with instructions for executing on a Torque cluster environment, and defaults to reading per-job resource specifications from ./cluster.json and run-specific configuration information from ./config.yaml. Currently, a small test dataset is included in ./example, and config.yaml is set up run the workflow on this test dataset.

However, unlike my previous shotgun workflow, this is meant to be a more modular process. Rather than executing the entire workflow all at once, there are a series of 'top level' rules defining particular sub-portions of the workflow. These include:

  • raw: initial read prep and quality description
  • qc: read qc, adapter trimming, host read removal
  • assemble: metagenome assembly
  • map: read mapping to assemblies for coverage profiling
  • bin: binning of genomes from metagenomes
  • anvio: creation Anvi'o binning visualizations (this requires a separate Conda environment)
  • taxonomy: taxonomic profiling of metagenomes

You can execute any of these top-level rules by specifying them after the launch command. Any required prerequisite rules will automatically be executed. For example:

bash launch.sh ./ raw will create links from your raw read files to the new data analysis folder, and run FastQC and MultiQC on the raw data.

bash launch.sh ./ qc will do the above, plus run adapter and quality trimming with Skewer, perform host read filtering, and run FastQC and MultiQC on the QC'd data.

This series of modules with rules to execute particular portions of a shotgun analysis workflow are located in the ./bin/snakefiles directory.

Per-run Configuration

Snakemake reads the information necessary to execute the workflow on a particular dataset from a yaml-format configuration file. Currently this includes information about rule-specific environment specification (this should be superceded by the in-built Snakemake environment handling noted above), parameter settings for specific rules (for example, trimming stringency), samples to use for particular steps (for example, only running assembly on a subset of samples), and filepath specification for samples.

An example configuration file is provided in config.yaml.

Currently, sample-specific information is passed in the config.yaml as a dictionary called samples, with each sample being keyed by a unique sample name. One record looks like this:

samples:
  sample1:
    filter_db: 
    forward: 
    - example/reads/Bs_L001_R1.fastq.gz
    - example/reads/Bs_L002_R1.fastq.gz
    reverse:
    - example/reads/Bs_L001_R2.fastq.gz
    - example/reads/Bs_L002_R2.fastq.gz

Each sample can have multiple forward and reverse read fastqs (for example, if the same sample was run across multiple lanes). These will be concatenated together prior to subsequent steps.

A sample-specific filter_db can also be specified -- this should be a Bowtie2 formatted mapping database filepath base. On Barnacle, you can access a few such reference dbs in my home directory:

/home/jgsanders/ref_data/genomes/mouse/mouse
/home/jgsanders/ref_data/genomes/Homo_sapiens_Bowtie2_v0.1/Homo_sapiens
/home/jgsanders/ref_data/genomes/phiX/phix

Usage notes

Snakemake requires that inputs and outputs be specified explicitly to construct the workflow graph. However, I have found that in practice there are samples in the sequencing manifest that do not show up in the demultiplexed sequence files, or do not yield all possible output files in Trimmomatic (for example, if there are no R1 reads that survive trimming). For this reason, I have been invoking this workflow with the --keep-going flag, which will run subsequent steps even if not all outputs are successfully generated.

Another parameter I've used successfully is --restart-times. Sometimes I've had jobs fail stochastically in the cluster environment, and setting this parameter to 1 (default is 0) will cause these failed jobs to automatically re-queue.

Finally, note that disk access-intensive steps are set to run on a temporary directory to allow execution on local scratch space in a cluster environment. This variable is called TMP_DIR_ROOT in the config.yaml, and should be set to the local scratch directory to enable this behavior.

How to run

On Barnacle, we want to avoid running compute-intensive jobs on the login node. That's what happens if we just run the included Snakefile without any additional information about how to access the cluster.

local execution (DON'T DO THIS):

snakemake --configfile config_Run1.yaml

Instead, I've provided a launch.sh script that is set up with some defaults chosen to improve execution on our cluster. Here's how you run it:

cluster execution (DO THIS):

bash launch.sh ./ --configfile config_Run1.yaml

Here's what's goingon behind the scenes in launch.sh to invoke the Snakemake workflow:

snakemake -j 16 \
--local-cores 4 \
-w 90 \
--max-jobs-per-second 8 \
--cluster-config cluster.json \
--cluster "qsub -k eo -m n -l nodes=1:ppn={cluster.n} -l mem={cluster.mem}gb -l walltime={cluster.time}" \
--directory "$@"

Let's go through what each of these parameters does.

-j 16: Runs no more than 16 jobs concurrently. If you have 96 samples that each need to get FastQC'd, it will only run 16 of these jobs at a time.

--local-cores 4: For rules specified as local rules (like linking files), limits to use of 4 CPUs at a time.

-w 90: Waits for at most 90 seconds after a job executes for the output files to be available. This has to do with tolerating latency on the filesystem: sometimes a file is created by a job but isn't immediately visible to the Snakemake process that's scheduling things.

--max-jobs-per-second 8: Limits the rate at which Snakemake is sending jobs to the cluster.

--cluster-config cluster.json Looks in the current directory for a file called cluster.json that contains information about how many resources to request from the cluster for each rule type.

--cluster "qsub -k eo [...]": This tells Snakemake how to send a job to the cluster scheduler, and how to request the specific resources defined in the cluster.json file.

--directory "$@": This passes all the input provided after bash launch.sh as further input to Snakemake. Because it comes right after the --directory flag, it's going to expect the first element of that input to be the path to the working directory where Snakemake should execute.

About

Modular snakemake workflow for assembly-based metagenomics

Resources

License

Stars

Watchers

Forks

Releases

No releases published

Packages

No packages published