Automated Library Preparation for the MCI Advantage Cancer Panel at Miami Cancer Institute Utilizing the Beckman Coulter Biomek i5 Span-8 Liquid Handler.

Abstract

MCI Advantage Panel is a next generation sequencing (NGS) assay based on TruSight Tumor 170 (Illumina). The panel is designed to detect genetic alterations [e.g. single nucleotide variants (SNVs), insertions and deletions (indels), amplifications (CNVs), and fusions/splice-variants (SVs)] in 170 cancer associated genes. Reference materials and patient samples (N=87) were utilized to establish the assay's performance characteristics. DNA/RNA extractions were performed from formalin fixed paraffin embedded (FFPE) samples using FormaPure Total (Beckman). Libraries were constructed with TruSight Tumor 170 kit on the Biomek i5 Span-8 NGS Workstation (Beckman). Sequencing is performed using Illumina NextSeq550 sequencers. Bioinformatic analysis is performed with the TST170 apps and Variant Interpreter on the HIPAA-compliant BaseSpace Enterprise platform. The sequence data were also analyzed and reports were generated with the Philips IntelliSpace Genomics platform. Of the 108 DNA libraries processed, exon coverage at 100X or greater was very high across all DNA libraries (mean 99.78% with a standard deviation of 0.09%). RNA sequencing metrics show that for the 80 samples sequenced median insert length varied somewhat more than the DNA libraries (mean 109bp with a standard deviation of 14bp). RNA Median CV Coverage at 1,000X was consistent (mean 0.53 with a standard deviation of 0.09). Analysis of 77 clinical FFPE samples showed 98.7% concordance with the orthogonal methods. Targeted NGS panel brings a unique opportunity for detection of multiple somatic alterations with a single platform in community cancer center setting. MCI Advantage Panel has an excellent overall coverage and shows high concordance with orthogonal methods.