Abstract 4089: A novel NGS-based sample tracking system for robust sample chain of custody and quality control in fresh frozen and FFPE tumor samples
Authors: Katherine Milligan, Yu Qiu, Andrea O'Hara, Carrie Ziemniak, David Corney, Haythem Latif
Journal: Cancer Research (2025)

Abstract When processing large cohorts of samples, sample identity and functional quality are key to ensure the derived data is accurate and attributed to the appropriate sample. This is especially critical when working with precious and degraded material such as formalin-fixed paraffin-embedded (FFPE) samples. A potential sample swap or poor sample quality may result in incorrect data or delays due to re-processing. While traditional methods such as qPCR can be used for quality control, they may not be an effective predictor of sample success in Next-Generation Sequencing (NGS) based assays due to differential susceptibility to inhibitors and interfering substances. Performing a functional quality control assessment using the same methodology for data generation allows for a more accurate prediction of performance as it would be similarly affected by interfering substances or sample degradation. We have created a scalable, cost-effective sample tracking and functional quality control assay that may be assessed prior to performing more expensive downstream sequencing assays. This assay was designed to confirm consistency in sample identity during processing and confirm identity when multiple samples are submitted from the same subject. This amplicon panel targets autosomal SNPs with varying minor allele frequencies in different ethnic populations, limiting a bias towards variation in European ethnic populations. The average minor allele frequency for the panel developed for the general population is 0.45, more specifically 0.46 for European background, 0.44 for African background and 0.46 for Asian background based on data from the Allele Frequency Aggregator (ALFA) database. Most amplicons were designed to be less than 150 base pairs to increase the likelihood of success for samples with degraded DNA. Targets were included on the X and Y chromosome for sex determination. Tumor-normal paired samples were processed on the sample tracking assay, targeting regions throughout the exome to allow tracking of sample identity. When comparing “normal” samples extracted from blood to their paired FFPE tumor tissue, known matches had an average of 98.0% concordance, while known mismatches had an average of 40.4% concordance, with the highest known mismatch showing 61.9% concordance. Additionally, when comparing well-characterized samples to their corresponding data from the 1000 Genomes Project, samples had 99.4% concordance. This exemplifies a robust sample tracking assay that can ensure sample identity is correct. A strong quality control assay enables NGS laboratories to identify poor sample quality or degradation, allowing for re-extraction or new sample collection before processing on a more expensive assay. This newly developed sample tracking assay ensures both sample integrity and identity, leading to an overall increase in data quality for cancer research studies. Citation Format: Katherine Milligan, Yu Qiu, Andrea O'Hara, Carrie Ziemniak, David Corney, Haythem Latif. A novel NGS-based sample tracking system for robust sample chain of custody and quality control in fresh frozen and FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4089.