This repository is obsolete as of June 2018. All the scripts and walkthroughs for 2bRAD genotyping, including reference-based, are now contained in the 2bRAD_denovo repository.
Mikhail Matz, matz@utexas.edu
2bRAD has been described in Wang et al 2012 http://www.nature.com/nmeth/journal/v9/n8/abs/nmeth.2023.html
2bRAD features a very simple library prep protocol with no intermediate purification stages. It involves triple barcode scheme, two being standard Illumina barcodes and one in-read ligated barcode for pooling libraries in 12-plexes midway through library prep to further minimize prep costs. 2bRAD generates 36-base tags that can be sequenced on either strand. In a full-representation version it results in one high-quality genotyped tag every 2.5-3.5 kb on average. With reduced-representation with modified ligated adapters, the number of genotyped tags can be reduced 4- or 16-fold, to save sequencing costs in applications not requiring dense genome sampling (such as population structure analysis or linkage mapping).
Unique features of 2bRAD bioinformatics are removal of PCR duplicates based on degenerate tag (allowing for 128-fold dynamic range of sequencing depth per allele), use of direct-reverse strand ratio as a quality fltering parameter, as well filtering and quality assessment based on genotyping replicates.
This collection of scripts is for reference-based genotyping with GATK pipeline, which works well even when mapping to genomes of different species.
This repository contains the lab protocol for sample preparation, as well as scripts and walkthrough (2bRAD_gatk_README.txt) for:
- trimming and quality filtering;
- removing PCR duplicates;
- mapping to reference genome with bowtie2;
- varint calling using UnifiedGenotyper
- recalibrating variant quality scores based on genotyping replicates;
- thinning and final filtering;
- quality assessment based on replicates.
Also included are walkthroughs for analysis (2brad_analysis.txt):
- principal component analysis and outlier detection using adegenet package in R
- computing Weir and Cockerham Fst
- BayeScan for Fst outlier detection and removal
- ADMIXTURE
IMPORTANT: Do not sequence 2bRAD libraries on a HiSeq 4000 lane alone! Invariant bases (adaptor, restriction site) will be read very poorly, resulting in read trimming issues. Mix 20% of PhiX libraries with your 2bRAD samples to avoid this problem, or share lane with someone else's non-2bRAD samples. (If you already did, no worries - there is a solution; contact me.)