Abstract 4897: Accurate variant detection using molecular barcodes

HaloPlex is an amplicon based method for targeted sequencing. The protocol utilizes specificity gained from restriction enzyme recognition, hybridization and DNA ligation to capture molecules originating from the target region to be sequenced. The target region is fully customizable from a single gene up to several thousand discrete regions. Amplicon based methods for multiplex target enrichment are, in general, convenient methods for capturing a wide range of target region sizes. However, in contrast to hybridization capture methods where random shearing is deployed, it is not possible for Haloplex or other amplicon based techniques to use the start point of paired end reads to identify duplicate reads. Duplicate read information can be useful for improving base calling accuracy and to monitor sampling to determine the degree of confidence to assign calls at different presumed allelic fractions. For somatic variants, which are generally present at a lower than 50% allelic fraction, it is even more advantageous to know how many molecules have been sampled when calling a particular base. To enable identification of duplicate reads from libraries prepared with Haloplex, we have added a molecular barcode to the introduced primer cassette. The molecular barcode consists of ten degenerate bases allowing for over one million unique sequences to be present for tagging of molecules. Using information derived from the molecular barcode sequences we demonstrate observation of variants down to 5% allelic fraction in multiple molecules tagged with different molecular barcodes. The new protocol has, besides the introduction of molecular barcodes, been optimized in a few additional aspects. Due to improved reagent formulations and streamlining of workflow, complete target enrichment can now be completed in less than 5 hours. Using 50 ng input we demonstrate >85% specificity and above 90% of target regions being covered at >10% of average depth. Citation Format: Charmian Cher, Henrik Johansson, Javelin Chi, Katie Zobeck, Linus Forsmark, Magnus Isaksson, Holly Hogrefe. Accurate variant detection using molecular barcodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4897. doi:10.1158/1538-7445.AM2015-4897