Tag: 2023

Automation of the Pillar Biosciences oncoReveal Solid Tumor 22 gene panel (ORST22) on the Biomek NGeniuS Next Generation Library Prep System

Pillar built and evaluated an automated application for its 48-gene oncoReveal® Solid Tumor v2 (oRSTv2) research-use panel on the Beckman Coulter Biomek NGeniuS library preparation system, taking DNA through to indexed libraries with minimal intervention. Twenty NA12878 replicates and 22 moderately formalin-compromised DNA samples at 19.35 ng input were processed across three runs of 24, 16, and 8 libraries, sequenced on Illumina NextSeq 550 and MiSeq with PiVAT® analysis. Every expected variant was called at its expected allele frequency — 478 of 478 across the study — with no cross-contamination in no-template controls.

Robust bioinformatic method for estimating tumor content in cfDNAusing denoised target sequencing

Copy number amplifications are the hardest alteration class to call from cell-free DNA, usually demanding high circulating tumor DNA and broad genomic coverage. Pillar built oncoCNA, a Bayesian non-parametric caller inside PiVAT®, and tested it on Seraseq® ctDNA reference material across allele frequencies from 0.25% to 5% at 10, 20 and 30 ng input on the 104-gene oncoReveal® Core LBx panel. Performance was comparable at 10 ng and 30 ng, and the choice of negative control mattered more than input amount — clinical normal samples gave 100% negative percent agreement where synthetic normals did not.

Detection of Microsatellite Instability (MSI) Using oncoReveal Core LBx, a Multiplexed, Single Tube, Liquid Biopsy Assay

Pillar added microsatellite instability (MSI) detection to the research-use oncoReveal® Core LBx liquid biopsy panel alongside SNV, indel and copy number calling, supporting immunotherapy selection — MSI-high tumors respond to checkpoint inhibitors, and the phenotype appears in roughly 15% of colorectal cancers and across Lynch syndrome. Twenty-seven amplicons covering 28 microsatellite sites were validated on 17 matched tumor-normal FFPE pairs, sequenced on Illumina NextSeq or MiSeq and scored in PiVAT® using MSIsensor. Sensitivity and specificity were 100% in both matched and unmatched calling modes, with correct calls holding down to 10% tumor dilution.

Robust detection of Copy Number Amplifications using Pillar’s oncoReveal™ Core LBx panel with low input from liquid biopsy samples

Copy number amplifications are the hardest alteration class to call from cell-free DNA, usually demanding high circulating tumor DNA and broad genomic coverage. Pillar built oncoCNA, a Bayesian non-parametric caller inside PiVAT®, and tested it on Seraseq® ctDNA reference material across allele frequencies from 0.25% to 5% at 10, 20 and 30 ng input on the 104-gene oncoReveal® Core LBx panel. Performance was comparable at 10 ng and 30 ng, and the choice of negative control mattered more than input amount — clinical normal samples gave 100% negative percent agreement where synthetic normals did not.

Robust Low Frequency Somatic Variant Detection in cfDNA Using a SLIMamp® Based liquid Biopsy Assay

Pillar assessed the oncoReveal® Core LBx panel — a UMI-free, 446-amplicon, 104-gene research-use liquid biopsy kit — for detecting low-frequency somatic variants in cell-free DNA. Seraseq® reference controls from 0.1% to 5% variant allele frequency at 10 and 30 ng input were run across 37 replicates alongside plasma from 16 cancer patients, two healthy donors and wild-type controls, sequenced on the Illumina NextSeq 550 with PiVAT® analysis. The panel detected 88% of expected variants at 0.25% VAF with 10 ng input and 96% with 30 ng, found high-priority variants down to 0.1%, and held per-position specificity above 99.99%.

oncoRevealTM Fusion LBx: Single-tube multiplexed PCR-based NGS assay for detection of multiple gene fusions from cell free RNA

Pillar developed oncoReveal® Fusion LBx, a research-use single-tube multiplexed PCR NGS assay detecting more than 150 fusion transcripts from cell-free RNA (cfRNA) — an analyte that avoids the long introns and repeat regions making fusions hard to catch in cell-free DNA. The panel targets breakpoints across 18 driver genes with 83 partners plus MET exon 14 skipping and EGFR variant III, and was assessed on Horizon and Seraseq® reference standards from 2.5 ng down to 0.025 ng RNA input on the Illumina MiSeq. Sensitivity and specificity were both 100% down to 0.25 ng input, equating to roughly 20 copies per target.