While lentiviral vectors have played a critical role in the emergence of gene-modified cell therapies, safety concerns remain regarding potential insertional mutagenesis. Regulatory authorities strongly recommend risk assessment and management of vector copy numbers (VCNs), integration profiles, and integration sites in the lentivirus-based cell and gene therapy products. However, accurately measuring these parameters remains a significant challenge due to the lack of standardized methodologies and VCN reference materials (RMs). Toward this challenge, we conducted an interlaboratory study on NIST candidate RMs for VCN measurements. The candidate RMs comprise five human genomic DNA samples or fixed cells from clonal Jurkat cell lines with defined VCNs ranging from 0 to 4. All 12 study participants were able to identify the VCN in the five blinded samples using quantitative PCR (qPCR), digital PCR (dPCR), or next generation sequencing (NGS) assays. Consensus value of VCN and integration sites in these candidate RMs were achieved. The fixed clonal VCN cells were also used to evaluate an emerging imaging-based technology called molecular combing. This interlaboratory assessment demonstrated the utility, commutability, and suitability of the NIST VCN candidate RMs for quality assurance and improved confidence in VCN, integration profile, and integration site measurements.

Long-read sequencing technologies enable resolution of structural variants (SV) and long-range genome assembly, but require high molecular weight (HMW) DNA of both high quantity and quality to produce optimal sequencing results. New DNA extraction methods have been developed but these have not been assessed for use in routine testing. The interlaboratory study described here tested four commonly used methods: Fire Monkey, Nanobind, Puregene and Genomic-tip with a reference cell line containing known chromosomal alterations. Samples were assessed with commonly applied approaches for evaluating DNA purity and integrity as well as a method based on linkage using digital PCR. Sequencing performance was evaluated and the impact of extraction method on structural variant calling investigated. All methods generally produced samples of acceptable purity although yield varied considerably between laboratories. Library preparation and sequencing were successful for all four methods, with Fire Monkey extracts achieving the highest N50 values, Genomic Tip giving the highest sequencing yields and Nanobind, the highest proportion of ultra-long reads (> 100 kb). The dPCR assay with duplexes at 100 kb and 150 kb distances was predictive of ultra-long reads and provides a more quantitative read-out (% linkage) than pulse-field gel electrophoresis (PFGE) which varied in performance between instruments and gel dyes. Neither PFGE nor dPCR were predictive of the proportion of short reads (< 10 kb). Coverage was a key factor in the success of SV calling, but this was dependent on SV caller. Megabase scale SVs were challenging to analyse with SV callers and required confirmation based on coverage plots and mapping of junction sequences, and the findings of earlier studies were only partially confirmed. This study highlights some of the challenges of HMW DNA extraction as well as the need for robust sample QC metrics to ensure optimal sequencing yield and read length which in turn influence the success of SV analysis. dPCR approaches for DNA integrity showed potential but require further development. As long-read methods are increasingly applied in routine settings such as clinical testing laboratories, cellular reference samples with well-characterised SVs are recommended as controls for the full long-read sequencing workflow.

Precision medicine approaches rely on accurate somatic variant detection, where the DNA input into genomic workflows is a key variable. However, there are no gold standard methods for total DNA quantification. In this study, a pentaplex reference gene panel using digital PCR (dPCR) was developed as a candidate reference method. The multiplex approach was compared between two assay chemistries, applied to healthy donor genomic DNA and plasma cell-free DNA (cfDNA) to measure the ERBB2 (HER2) copy number variation in cancer cell line DNA. The multiplex approach demonstrated robust performance with the two assay chemistries, demonstrating comparable results and a wide dynamic range. Ratios of reference genes were close to the expected 1:1 in healthy samples; however, some small but significant differences (<1.2-fold) were observed in one of the five targets. Expanded relative measurement uncertainty was 12.1–19.8% for healthy gDNA and 9.2–25.2% for cfDNA. The multiplex approach afforded lower measurement uncertainty compared to the use of a single reference for total DNA quantification, which is an advantage for its potential use as a calibration method. It avoided potential biases in the application to CNV quantification of cancer samples, where cancer genome instability may be prominent.