Plasmid-Based Materials as Multiplex Quality Controls and Calibrators for Clinical Next-Generation Sequencing Assays.
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.
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
David J
DJ
Sims
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Robin D
RD
Harrington
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
Eric C
EC
Polley
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Thomas D
TD
Forbes
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Michele G
MG
Mehaffey
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Paul M
PM
McGregor
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Corinne E
CE
Camalier
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Kneshay N
KN
Harper
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Courtney H
CH
Bouk
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
Biswajit
B
Das
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
Barbara A
BA
Conley
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
James H
JH
Doroshow
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
P Mickey
PM
Williams
Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland. Electronic address: jason.lih@nih.gov.
Chih-Jian
CJ
Lih
D019295:Computational Biology / Q000379:methods; D058893:DNA Barcoding, Taxonomic / Q000379:methods / Q000592:standards; D005820:Genetic Testing / Q000379:methods* / Q000592:standards*; D023281:Genomics / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D057188:Workflow