Category: Assay Development and Screening
Genotyping by Sequencing (GBS) has become an invaluable tool in animal and plant genomics to identify disease susceptibility, favorable traits, and determine parentage. GBS, performed on Next Generation Sequencing (NGS) platforms, typically uses either restriction enzyme-mediated techniques or amplicon re-sequencing to cost-effectively generate a large amount of data. GBS techniques require the input DNA sample be amplified and otherwise treated to generate a library of amplicons with universal priming sites on their ends. This library preparation, a key part of the GBS workflow, is inherently laborious, time consuming, and expensive. We have developed a high throughput library preparation workflow for amplicon libraries that utilizes half the reaction volume of traditional library preparation workflows and significantly decreases costs. AgriSeq library preparation technology is a powerful, flexible and customizable amplicon re-sequencing workflow, utilizing ultra-high multiplex PCR for targeted sequencing of known SNPs, MNPs, and INDELs. This high throughput library preparation workflow is capable of processing hundreds to thousands of samples per day, with sequencing results in as little as two days. The highly-repetitive nature of handling such a large number of samples makes it an excellent prospect for automation. Automation of AgriSeq library preparation workflow can be conducted on most any open liquid handling platform reducing variability, increasing consistency and reproducibility. By incorporating a pooling step that allows the bead-based clean-up from up to four 384-well plates to be completed on a single 96-well plate, the workflow results in significant time savings and reduces costs. Performance of the AgriSeq workflow was validated with an amplicon panel designed to target all the SNPs designated by the Bovine ISAG SNP Parentage Panel (2013). This panel was tested with a diverse set of bovine DNA samples on multiple liquid handling platforms. 384-barcoded samples were processed with the high-throughput library prep automation workflow and a manual 96-well workflow and sequenced on the Ion S5 Sequencing System. Equivalent performance was achieved between the automated and manual workflows including genotyping call rate, mean coverage depth, and coverage uniformity. Additionally, the automated workflow demonstrates high intra-run and inter-run genotype concordance. This library preparation workflow provides an automatable, fast and economical alternative to larger volume, lower throughput library prep methods without sacrificing performance.
Roy Willis– Staff Scientist, Thermo Fisher Scientific, Austin, TX