Simultaneous Detection of Single Nucleotide Variants and Copy Number Variations in Expanded Carrier Screen Using Next-generation Sequencing


Expanded carrier screening (ECS) is a testing approach that involves screening for many genetic conditions without regard to ethnicity. With recent advances in next-generation sequencing (NGS) technology and bioinformatics techniques, ECS has become a feasible and cost-effective approach for evaluating the carrier status of couples considering pregnancy. We established a custom ECS panel of 220 genes that is capable of detecting not only single nucleotide variants (SNVs) and small insertions/deletions (indels), but also copy number variations (CNVs) using NGS coupled with an advanced bioinformatics pipeline and tools developed in-house.


DNA was isolated from whole blood and saliva samples and then enriched for targeted regions using PCR with
specific primer sets to create DNA libraries. A custom QIAGEN QIAseq Targeted DNA Panel was used for the library preparation and Hamilton Microlab STAR instruments were used to automate the process. Prepared libraries were then loaded on an Illumina NextSeq 500 and sequenced at 2 x 150 bp reads. Data generated by the sequencer was processed usingĀ a custom bioinformatics pipeline developed at True Health for molecular tagged sequencing reads. SNVs and small indels were called using FreeBayes and CNV analysis was performed using BioDiscovery Nexus
Copy Number software.


Our ECS panel of 220 genes was designed based on ACMG and ACOG guidelines. The panel covers the
exonic regions and common hotspots associated with genetic conditions in the sequenced genes. Characterized specimens (n=82) were interrogated for known variants reported in literature or identified through sequencing assays performed by CLIA-certified reference laboratories. Minimum mean sequencing coverage for the panel is 400X and base positions were evaluated for sequencing variants if they achieved the required 50X minimum coverage for variant calling. High coverage uniformity was observed across the targeted bases. Accuracy was found
to be 99.99% for SNVs and small indels and 100% for CNVs. Analytical specificity was determined by sequencing the NIST GIAB reference standards NA12878 and NA24385 and specimens with positive CNVs previously confirmed by an independent reference laboratory. Specificity was found to be 100% for SNVs and small indels and 99.65% for CNVs. Precision was found to be 100% for SNVs, small indels, and CNVs. The sensitivity/limit of detection study showed variant detectability down to 10% allele frequency.


Based on the results of this study, the custom ECS panel was validated for patient testing in our CLIAcertified laboratory. This ECS panel has the advantage of being able to detect SNVs, indels, and CNVs with a single NGS assay.

P. Hetterich, M. Ta, T. Lewis, D. Taylor, A. Patrick, M. Saniepay, A. Barry, J. Quan, A. Jeffers-Brown, B. Sutton, G. Smith, W. Xu
True Health Diagnostics, Richmond, VA.

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