Abstract 3532: Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow

• Published in: Cancer research (Chicago, Ill.) Ill.), 2019-07, Vol.79 (13_Supplement), p.3532-3532
• Main Authors: Pendleton, Kathryn E., Liu, Yang L., Lin, Lifeng, Lee, Lucie S., Liu, Jeffery, Liu, Guoying, Liu, Zhitong
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2019-3532

Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB. Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics’ variant calling algorithm. Using the CleanPlex technology, this prototype TMB panel exhibits >95% uniformity at 0.2X mean, limited GC bias, and >94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours. Citation Format: Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. Tumor mutational burden (TMB) assessment a