Snakemake Barcoding Assembly Pipeline

Pipeline for processing Illumina sequencing data consisting of AAFC Diptera COI PCR amplicons. Current release is not compatible with other multi-fragment COI strategies.

1. Trims adapters and bases below 10 quality score BBDuk
2. Merges paired end reads into either Fragment A or Fragment B corresponding to PCR amplicons
3. Removes degenerate primers from the fragments
4. Dereplicates merged reads using VSEARCH VSearch
5. Merges top two merged reads using Emboss Merger Emboss
6. Evaluates results and generates a multi-fasta

Prerequisites

• Conda Installation
• Snakemake Installation
• Git

Installing Miniconda + Snakemake

wget https://repo.continuum.io/miniconda/Miniconda3-latest-Linux-x86_64.sh
bash Miniconda3-latest-Linux-x86_64.sh
conda install -c bioconda -c conda-forge snakemake

Getting Started

• Create and enter a working directory:
• Clone repository

git clone https://github.com/AAFC-BICoE/snakemake-barcoding-assembly-pipeline.git .

• Create a folder named "fastq" and populate with COI Illumina reads in fastq.gz format
• Initialize conda environment containing snakemake

source ~/miniconda3/bin/activate

• Invoke pipeline from within working directory. Adjust cores to suit computer

snakemake --use-conda -k --cores 32

• Alternative pipeline to map reads to COI reference gene

snakemake -s barcoding_snakefile --use-conda -k --cores 32

Methodology

Pipeline was designed to handle COI genes amplified in overlapping fragments from thousands of Diptera specimens.

Reads are trimmed of adaptors and poor quality bases. Paired end reads are merged into single long fragments. Each fragment is examined for primers, specifically Fragment A forward primer, and Fragment B reverse primer. If Fragment A forward primer is detected, read is trimmed of all Fragment A primers. If Fragment B reverse primer is detected, read is trimmed of all Fragment B primers. Reads are then de-replicated and abundance calculated. Ideal scenario is two very high abundance reads representing Fragment A and B, with minimal or no other reads. The top two reads are merged together to form a single CO1 sequence.

Curated CO1 sequences that contain <10% contamination/error reads, have a minimum of 40 combined reads that pass all filtering steps, and are the correct 646 bp length are placed in Curated_Barcodes.fasta Quality checks for all sequences are listed in Summary_Output.csv

Built With

• Python - Programming language
• Conda - Package, dependency and environment management
• Snakemake - Workflow management system
• BioPython - Tools for biological computation

Copyright

Government of Canada, Agriculture & Agri-Food Canada

License

This project is licensed under the MIT License - see the LICENSE file for details

Citations

• Snakemake
  Köster, Johannes and Rahmann, Sven. “Snakemake - A scalable bioinformatics workflow engine”. Bioinformatics 2012.

• Bold Retriever
  Vesterinen, E. J., Ruokolainen, L., Wahlberg, N., Peña, C., Roslin, T., Laine, V. N., Vasko, V., Sääksjärvi, I. E., Norrdahl, K., and Lilley, T. M. (2016) What you need is what you eat? Prey selection by the bat Myotis daubentonii. Molecular Ecology, 25(7), 1581–1594. doi:10.1111/mec.13564

• BBTools
  Brian-JGI (2018) BBTools is a suite of fast, multithreaded bioinformatics tools designed for analysis of DNA and RNA sequence data.https://jgi.doe.gov/data-and-tools/bbtools/

• BOLD
  Ratnasingham, S. & Hebert, P. D. N. (2007). BOLD : The Barcode of Life Data System (www.barcodinglife.org). Molecular Ecology Notes 7, 355–364. DOI: 10.1111/j.1471-8286.2006.01678.x

• VSearch
  Rognes T, Flouri T, Nichols B, Quince C, Mahé F. (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. doi: 10.7717/peerj.2584

• Emboss
  Rice P., Longden I. and Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics. 2000 16(6):276-277

Author

Jackson Eyres
Bioinformatics Programmer
Agriculture & Agri-Food Canada
jackson.eyres@canada.ca