Validation of the GeneReader NGS system for BRCA 1 and BRCA 2 genotyping

Background: BRCA1/2 testing with NGS technology offers many advantages over Sanger sequencing, including high throughput, and automated analysis. However, multiple barriers still exist for its broader adoption in clinical laboratory, such as fragmented workflow and complex bioinformatics analysis. GeneReader NGS system includes all sample processing steps starting from nucleic acid extraction to an integrated bioinformatics solution that allows for direct access to real-time updates from clinical knowledge and evidence. We evaluated performance of the GeneReader NGS Platform for BRCA 1 and BRCA 2 genotyping.
Methods: BRCA1 and BRCA2 sequence determination using GeneReader was performed in a total of 100 patients, who were diagnosed breast or ovarian cancer and previously tested BRCA1 and BRCA2 analysis using Sanger sequencing. Libraries (253 amplicons covering 37.5 kb) were prepared using QIAGEN Library Kit. Emulsion PCR and bead enrichment steps were performed using GeneReader QIAcube and Clonal Amp Q Kit. All the procedures were performed according to manufacturer’s protocols. QCI Analyze software performed read alignment, quality control, variant calling, and clinical report generation. The results generated by GeneReader were compared to those obtained by Sanger sequencing. For reproducibility of variant calling, we performed the process in triplicate using 28 samples and analyzed the results. We reviewed turnaround time (TAT) of total process and its hands on time.
Results: Greater than 99.9% of the target regions showed read depths >100x and the percentage of reads in on-target regions was 90.5%. Variants generated from GeneReader achieved 100% (13/13) and 98.9% (722/730) concordance with those generated from Sanger sequencing in insertion/deletions (indels) and single nucleotide variations (SNVs), relatively. The annotation of the GeneReader system showed over 99.9% (744/745) concordance with databases in ClinVar. All of 272 SNVs and 7 indel variants showed 100% calling reproducibility. The GeneReader provided a truly integrated workflow and most process was composed of hands off time (3250 min, 87% of total TAT).
Conclusions: The GeneReader NGS system for BRCA 1 and BRCA 2 genotyping showed great performance and takes much shorter hands on time compared with Sanger sequencing. With a full end-to-end solution with integrated sample preparation to bioinformatics interpretation, the GeneReader NGS System can be useful for clinical laboratory practices.

Lee, N. Park; W. Lee, S. Chun, W. Min

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