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Evaluation of a Targeted NGS Panel Using Single-Vial Amplification of Candidate Genes in Solid Tumors

Poster presented by Helen Fernandes

Devon Hemnauth1, Subit Barua1, Erin Petrilli2, Chris Freeman1, Susan Hsiao1, Mahesh Mansukhani1 & Helen Fernandes1
1Laboratory of Personalized Genomic Medicine, Department of Pathology and Cell Biology, Columbia University Medical Center, New York; 2Pillar Biosciences, Natick, MA.

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Introduction The use of targeted Next Generation Sequencing (NGS) assays for detection of variants with therapeutic, diagnostic and prognostic potential is well established. Recently, the association of variants in genes leading to DNA damage repair deficiency with response to immunotherapy has expanded the utility of NGS panels. We customized a 47-gene panel using single-tube Stem-Loop Inhibition-Mediated Amplification (SLIMampTM) technology (Pillar Biosciences) for detection of informative variants in tumors including, but not limited to, NSCLC, colorectal and pancreatic cancer, GIST, melanomas, gliomas and thyroid tumors. The 24kb panel covered hotspots in most genes and entire CDS in 3 genes with oncogenic potential. Robust performance with minimal DNA input and limited neoplastic material was central to the experimental design.

Methods Accuracy, precision, reproducibility and sensitivity were assessed using 110 patient samples and 15 controls. The assay was challenged with total DNA input of 2-3ng obtained from tumors with 10-20% of neoplastic cells. The variant allelic fractions of the samples interrogated ranged from 2.5% to >80%. Specificity was checked using GIAB NA12878. Quality Control was monitored using a engineered control with multiple targeted variants having VAF ranging from 4%-15%. For each run, up to 24 samples were normalized, pooled and run using the MiSeq reagent kit V2 (Illumina). Data analysis including sequence alignment, variant calling and annotation was performed using FASTQ files with the Pillar Variant Analysis Toolkit (PiVAT).

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Additional Pillar Resources

Making Precision Medicine Accessible with SLIMamp®: Robust, Rapid, Single-Tube Multiplex NGS Panels

Presented by Dr. Martin Zillmann, Chief Operating Officer and Vice President of Research and Development at Pillar Biosciences at The Association for Biomolecular Research Facilities (ABRF) 2019 Annual Meeting

Dr. Zillmann presented data demonstrating the performance of Pillar’s key proprietary NGS technologies, including SLIMamp® chemistry, ampPD™ primer design software, and PiVAT® bioinformatics pipeline software. Dr. Zillmann also presented data from de-identified clinical samples demonstrating high sensitivity for the detection of low allele frequency mutations from low DNA inputs.

Pillar’s robust amplicon-based workflow is simple and robust. SLIMamp enables library preparation via tiled (overlapping) amplicons in a single-tube. SLIMamp and PiVAT allow for accurate detection even from poor-quality FFPE samples. Pillar’s capabilities were highlighted by a case study detailing an Ivy league-affiliated cancer institution’s use of multiple custom and standard pillar NGS panels.