Plasmid-Based Materials as Multiplex Quality Controls and Calibrators for Clinical Next-Generation Sequencing Assays

Abstract

Although next-generation sequencing technologies have been widely adapted for clinical diagnostic applications, an urgent need exists for multianalyte calibrator materials and controls to evaluate the performance of these assays. Control materials will also play a major role in the assessment, development, and selection of appropriate alignment and variant calling pipelines. We report an approach to provide effective multianalyte controls for next-generation sequencing assays, referred to as the control plasmid spiked-in genome (CPSG). Control plasmids that contain approximately 1000 bases of human genomic sequence with a specific mutation of interest positioned near the middle of the insert and a nearby 6-bp molecular barcode were synthesized, linearized, quantitated, and spiked into genomic DNA derived from formalin-fixed, paraffin-embedded-prepared hapmap cell lines at defined copy number ratios. Serial titration experiments demonstrated the CPSGs performed with similar efficiency of variant detection as formalin-fixed, paraffin-embedded cell line genomic DNA. Repetitive analyses of one lot of CPSGs 90 times during 18 months revealed that the reagents were stable with consistent detection of each of the plasmids at similar variant allele frequencies. CPSGs are designed to work across most next-generation sequencing methods, platforms, and data analysis pipelines. CPSGs are robust controls and can be used to evaluate the performance of different next-generation sequencing diagnostic assays, assess data analysis pipelines, and ensure robust assay performance metrics.

An interlaboratory study of complex variant detection

“…Our study did not consider issues that arise in somatic testing even though the genes we investigated are included on many somatic panels and the variants we studied could influence therapeutic selection for PARP inhibitors or immunotherapies. A similar SRS for somatic tests could be constructed by titrating the same plasmids to a low variant allele fraction (VAF) as has been done previously [12][13][14]. The combined effect of reference bias (Figure 1f) with a low starting VAF would likely uncover additional sensitivity limitations for the medium and large indels, an important issue to explore further.…”

“…In this interlaboratory study, we explored one potential solution to this problem: the creation of a synthetic reference sample (SRS) in which multiple technically challenging variants are synthesized and then introduced into a known human genomic background. This technique has previously been used in analyses of both germline and somatic panel tests [12][13][14], but these studies did not include many variants of high technical complexity. An approach using synthetic exogenous sequences has also been developed [15] but cannot be used with existing panel or exome tests.…”

The current state of molecular testing in the treatment of patients with solid tumors, 2019

“…However, the more important parameters for determining effective, personalized treatment for patients are accuracy, reproducibility, and sensitivity of the assay, and these can be much more challenging to critically evaluate but must be rigorously validated . The use of a highly multiplexed, consistent, and well‐characterized reference material greatly facilitates the comparison of assay systems …”

“…[173][174][175][176] The use of a highly multiplexed, consistent, and well-characterized reference material greatly facilitates the comparison of assay systems. 177…”

Development and Characterization of Reference Materials for Genetic Testing: Focus on Public Partnerships

“…In yet unpublished data, a set of plasmids using the same plasmid backbone was generated, and can be easily manufactured with clonal purity [42]. Each plasmid consists of single known variant which is described in the Catalogue of Somatic Mutations in Cancer (COSMIC) database, located approximately in the middle of a 1 kb DNA fragment [43].…”