16S rRNA gene amplicon sequencing pipeline using DADA2, implemented in Nextflow
A dada2-based workflow using the Nextflow workflow manager. The basic pipeline is currently implemented, including some basic read-tracking. This pipeline is adapted from https://github.com/HPCBio/dada2-Nextflow for implementation on the UCT high-performance compute cluster
Basic usage:
This pipeline can be run specifying parameters in a config file or with command line flags.
The typical example for running the pipeline with command line flags is as follows:
nextflow run uct-cbio/16S-rDNA-dada2-pipeline --reads '*_R{1,2}.fastq.gz' --trimFor 24 --trimRev 25 --reference 'gg_13_8_train_set_97.fa.gz' -profile uct_hex
The typical command for running the pipeline with command line flags is as follows:
nextflow run -c <dada2.conf> <dada2.nf> -profile uct_hext
where:
dada2.conf is the configuration file
dada2.nf is the pipeline script
To override existing values from the command line, please type these parameters:
Mandatory arguments:
--reads Path to input data (must be surrounded with quotes)
-profile Hardware config to use. Currently profile available for UCT's HPC 'uct_hex' - create your own if necessary
NB -profile should always be specified on the command line, not in the config file
--trimFor integer. headcrop of read1 (set 0 if no trimming is needed)
--trimRev integer. headcrop of read2 (set 0 if no trimming is needed)
--reference Path to taxonomic database to be used for annotation (e.g. gg_13_8_train_set_97.fa.gz)
All available read preparation parameters:
--trimFor integer. headcrop of read1
--trimRev integer. headcrop of read2
--truncFor integer. tailcrop of read1. enforced before trimming
--truncRev integer. tailcrop of read2. enforced before trimming
--maxEEFor integer. After truncation, R1 reads with higher than maxEE "expected errors" will be discarded. EE = sum(10^(-Q/10)), default=2
--maxEERev integer. After truncation, R1 reads with higher than maxEE "expected errors" will be discarded. EE = sum(10^(-Q/10)), default=2
--truncQ integer. Truncate reads at the first instance of a quality score less than or equal to truncQ; default=2
--maxN integer. Discard reads with more than maxN number of Ns in read; default=0
--maxLen integer. maximum length of sequence; maxLen is enforced before trimming and truncation; default=Inf (no maximum)
--minLen integer. minLen is enforced after trimming and truncation; default=50
--rmPhiX {"T","F"}. remove PhiX from read
--minOverlap integer. minimum length of the overlap required for merging R1 and R2; default=20 (dada2 package default=12)
--maxMismatch integer. The maximum mismatches allowed in the overlap region; default=0
--trimOverhang {"T","F"}. If "T" (true), "overhangs" in the alignment between R1 and R2 are trimmed off.
"Overhangs" are when R2 extends past the start of R1, and vice-versa, as can happen when reads are longer than the amplicon and read into the other-direction primer region. Default="F" (false)
Other arguments:
--pool Should sample pooling be used to aid identification of low-abundance ASVs? Options are
pseudo pooling: "pseudo", true: "T", false: "F"
--outdir The output directory where the results will be saved
--email Set this parameter to your e-mail address to get a summary e-mail with details of the run
sent to you when the workflow exits
-name Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic.
Help:
--help Will print out summary above when executing nextflow run uct-cbio/16S-rDNA-dada2-pipeline
Merging arguments (optional):
--minOverlap The minimum length of the overlap required for merging R1 and R2; default=20 (dada2 package default=12)
--maxMismatch The maximum mismatches allowed in the overlap region; default=0.
--trimOverhang If "T" (true), "overhangs" in the alignment between R1 and R2 are trimmed off. "Overhangs" are when R2 extends past the start of R1, and vice-versa, as can happen
when reads are longer than the amplicon and read into the other-direction primer region. Default="F" (false)
Taxonomic arguments (optional):
--species Specify path to fasta file. See dada2 addSpecies() for more detail. Example run:
To run on UCT hex
1) Start a 'screen' session from the headnode
2) Start an interactive job using: qsub -I -q UCTlong -l nodes=1:series600:ppn=1 -d `pwd`
3) A typical command would look something like:
nextflow run uct-cbio/16S-rDNA-dada2-pipeline --trimFor 24 --trimRev 25 --reference /specify/relevant/directory/gg_13_8_train_set_97.fa.gz --email katieviljoen@gmail.com -profile uct_hex --reads '/specify/relevant/directory/*{R1,R2}.fastq' -with-singularity /scratch/DB/bio/singularity-containers/1a32017e5935-2018-05-31- db3a9cebe9fc.img --pool 'pseudo'Prerequisites
Nextflow, dada2 (>= 1.8), R (>= 3.2.0), Rcpp (>= 0.11.2), methods (>= 3.2.0), DECIPHER, phangorn, biomformat Note: if you are working on UCT hex you can simply use the singularity image specified in the uct_hex profile (no need to install these R packages)
Documentation
The uct-cbio/16S-rDNA-dada2-pipeline pipeline comes with documentation about the pipeline, found in the docs/ directory:
Built With
Credits
The initial implementation of the DADA2 pipeline as a Nextflow workflow (https://github.com/HPCBio/dada2-Nextflow) was done by Chris Fields from the high performance computational biology unit at the University of Illinois (http://www.hpcbio.illinois.edu). Please remember to cite the authors of DADA2 when using this pipeline. Further development to the Nextflow workflow and containerisation in Docker and Singularity for implementation on UCT's HPC was done by Dr Katie Lennard and Gerrit Botha, with inspiration and code snippets from Phil Ewels http://nf-co.re/
License
This project is licensed under the MIT License - see the LICENSE.md file for details