Implementation of a High Throughput AgriSeqTM Workflow for GBS

Genotyping by Sequencing (GBS) has become an invaluable tool in animal and plant genomics to identify disease susceptibility, favorable traits, or even determine parentage. GBS is typically performed on Next Generation Sequencing (NGS) platforms using either restriction enzyme-mediated techniques or through amplicon re-sequencing. AgriSeq^TM library prep is a powerful, flexible and customizable amplicon re-sequencing workflow which utilizes ultra-high multiplex PCR for targeted sequencing of known SNPs, MNPs, and INDELs.

Library preparation is one of the most costly and time consuming steps in the sequencing process. We have developed a 384-well library prep workflow for amplicon libraries that utilizes half the reaction volume of traditional 96-well workflows. Decreasing the reaction volume and increasing library throughput significantly decrease workflow costs. We have incorporated a pooling step into the workflow that allows the bead-based clean-up from up to four 384-well plates to be completed on a single 96-well plate resulting in significant time savings. Library processing time was reduced by >1 hour with >50% reagent usage.

Performance of the high throughput AgriSeq library prep workflow was validated with multiple Ion AmpliSeq^TM panels. 384 barcoded samples were processed with the high-throughput AgriSeq library prep workflow and a standard volume 96-well workflow and sequenced on the Ion S5^TM XL System. Equivalent performance was achieved between the two workflows including genotyping call rate, mean coverage depth, and coverage uniformity. The high throughput AgriSeq library prep workflow provides a fast and economical alternative to larger volume, lower throughput library prep methods without sacrificing performance.