Abstract 324: Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples
Authors: Rock Rachel , Prasad Nripesh , Robinson Melanie , Wildman Dinnen , Montoya Olivia , Sykes Michael , Umylyn Boris , Halsey Thomas 
Published: March 2024

FFPE is the most common method used to store precious clinical samples at room temperature for extended periods (sometimes&gt;15 years). FFPE samples are therefore one of the most commonly available samples that can be used for discovery and diagnosis. Whole Exome Sequencing (WES) is one of the most used NGS assays that is used for profiling somatic and tumor mutations. With future clinical trials moving towards less invasive collection methodology, core needle biopsy samples are becoming a method of choice. The FFPE fixation process leads to the degradation of nucleic acids due to cross-linkage and the availability of sufficient nucleic acid from CNB fixed samples for downstream NGS assays is becoming limited. Here we report a comparison of two Whole Exome capture kits from several degraded FFPE samples, NA12878, OncoSpan gDNA HD827 and SeraSeq Breast CNV mix gDNA samples using the xGen Exome Hyb Panel v2 (IDT) Kit and the Twist Exome 2.0 (TWIST) Kit. Libraries were prepared in triplicate at 50ng, and 10ng using the Ultra™ II DNA Library Prep (NEB) kit. Pre-capture libraries were evaluated to determine the total DNA library available for capture using the KAPA Library Quantification Kit (Roche) kit. The recommendations for input amount vary between the kits, with IDT recommending 500ng, and TWIST recommending 200ng per library. KAPA Library Quantification Kit kit was used to assess the nanomolar amounts of ligated libraries. Final libraries were pooled equimolar and then sequenced as 2 × 100bp Paired-end sequencing on the NovaSeq 6000 instrument to achieve a target of 150-300X overall coverage. The performance of each capture kit was evaluated on the number of libraries eligible for capture, percent duplication rate, median insert size, and mean target coverage. IDT capture had 49% of libraries eligible at recommended input of 500ng, with 34% of libraries generating between 100-499ng input and the remaining 17% failing to generate 100ng input for hybridization. TWIST capture had 83% of libraries eligible at recommended input of 200ng, with 4% of libraries generating between 50-199ng input and the remaining 13% failing to generate 50ng input for hybridization. Most notably, 96% of libraries at 50ng DNA input were eligible for TWIST capture, while only 46% of libraries at 50 ng DNA input were eligible for IDT capture. The average Q30 for IDT and TWIST capture were 93-94% respectively. Both IDT and TWIST capture kits produced similar duplication rates and average mean target coverage was comparable. Taken together, our testing shows that TWIST and IDT kits generated comparable sequencing metrics. However, the TWIST Kit outperforms the IDT Kit in percent eligible libraries qualifying for hybridization capture. TWIST Kit is a great qualifier for highly degraded FFPE samples, especially those with limited input DNA material.

Citation Format: Rachel Rock, Nripesh Prasad, Melanie Robinson, Dinnen Wildman, Olivia Montoya, Michael Sykes, Boris Umylyn, Thomas Halsey. Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 324.