Abstract

High Sensitivity Variant Detection with SureSelectXT HS Library Prep optimized for FFPE

Anuj Gupta, Senior Scientist, Agilent Technologies

Advancements in NGS and targetenrichment have made it possible todetect low-occurrence mutations in aheterogeneous mixture. However, furtheradvancements are hindered by systemicerrors in PCR and sequencing methods, which introduce bias in the form of duplicatesand associated non-uniform amplificationand artifacts that generate sequence changes notpresent in the original sample. Sensitivity refers to the percentage of true variants that are correctly identified. In most cases, sensitivity is limited by the input amount, performance of the assay, and PCR and sequencing error rates. As a result, the error level seen with traditional NGS assays typically interferes with the confidence of detecting low frequency variants.This issue is especially relevant for more heterogeneous cell populations, such as certain tumor subpopulations.Agilent SureSelectXT HS incorporates molecular barcodes, which enables users to filter out artifacts produced during library preparation, target enrichment and sequencing that cause false positive variants. Attachment of the molecular barcodes to a DNA fragment results in a unique identifier assigned to each input molecule.Molecular barcoding with SureSelectXT HS achieves industry-leading metrics, allowing the detection of rare variants down to = 1%. Molecularbarcodes can enhance confidence,particularly in clinical research sampleswherein the mutation prevalence can be inthe same range as the error rate.SureSelectXT HS produces more complex libraries with a higher percentage of reads in targeted regions for a wide range of tissue types—in both fresh and FFPE samples—with only 10 - 200 ng of input material. To improve single nucleotide variant (SNV) calling and consistency across FFPE samples of varying quality, Agilent has developed workflow solution that features DNA pre-qualification and one-tube library preparation.SureSelectXT HS reduces the number of individual enzymatic steps, clean-up steps and sample transfers, allowing users to streamline and maintain library complexity, especially at lower input amounts (as little as 10 ng). The 90-minute hybridization, combined with master-mixed reagents, enables users to go from samples to sequencing-ready libraries in one day.