How to construct DNA libraries using Genotyping-by-Sequencing (GBS)/ddRADseq

Genotyping-by-Sequencing (GBS) is a method for processing genomic DNA into libraries for next-generation sequencing. GBS is commonly used by plant geneticists because it makes sequencing more high-throughput and affordable. Examples of downstream applications include genetic linkage mapping, genome-wide association studies (GWAS), genomic diversity and association studies, molecular marker discovery and genomic selection (GS). This video specifically uses the double digest restriction-site associated DNA sequencing (ddRADseq) pipeline as it has been demonstrated to work on sweet basil. Although the workflow is typically automated in specialized genomics labs, a more manual workflow is presented for broader accessibility and teaching purposes. Each step of the pipeline is listed below with its time stop: Background 00:13 What is ddRADseq? 00:28 Why do we use ddRADseq? 00:56 Step 1: gDNA dilution 1:57 Step 2: digest 5:02 Step 3: ligation 7:05 Step 4: clean up DNA 11:14 Step 5: PCR 18:44 Step 6: qubit 20:44 Step 7: pooling 25:36 Acknowledgements 27:07 Citations: Poland et al. 2012- https://journals.plos.org/plosone/art... Pyne et al. 2017- https://journals.plos.org/plosone/art... For more information on the Simon lab and RU basil, visit: Website: https://newuseag.rutgers.edu/ Instagram: @rutgersbasil This video was produced by Lara Brindisi, PhD Candidate at Rutgers University Email: [email protected]