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Pillar Biosciences at
Association for Molecular Pathology's Annual Meeting and Expo 2019

Pillar® NGS assays provide versatility across platforms, mutation types and panel designs.

Corporate Workshop | Watch video

Finding flexibility in a resource limited world: Considerations for amplicon-based NGS enrichment in laboratories running multiple sample and assay types

Presented on November 6, 2019

Description
While there are multiple advantages to implementing NGS in the clinical lab, NGS testing has the potential to be both time and resource consuming. Cynthia Schandl, MD, PhD and Julie Hirschhorn, PhD of the Medical University of South Carolina will discuss their process for selection of solid tumor and myeloid panels for evaluation. While keeping clinical relevance in mind, the selection process involved assessing assays for flexibility, affordability, and ease-of-use. This presentation will include an evaluation and discussion of the single-tube Stem-Loop Inhibition Mediated Amplification (SLIMamp®) technology from Pillar Biosciences for detection of variants in solid and myeloid tumors.

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Detection of Internal Tandem Duplications in the FLT3 gene using Pillar PiVAT Software

Introduction:
The FLT3 gene is one of the most important genes in myeloid cancers and also one of the most difficult to sequence accurately due to the presence of internal tandem duplications (ITDs). Detection of ITDs is challenging for many next-generation sequencing (NGS) bioinformatics pipelines due to limited read lengths. We developed a novel method to reliably and accurately detect ITDs and incorporated it into the Pillar Variant Analysis Toolkit (PiVAT®).

Methods:
To detect ITDs, we utilized several components within PiVAT, including a proprietary paired-end assembly method, local realignment, and processing of SA tag information. To assess the performance of PiVAT, publicly available data from 208 clinically detected ITDs identified in a 664-patient acute myelogenous leukemia (AML) study was used to simulate paired-end read NGS data for analysis. The data contained ITDs ranging in length from 3bp to 201bp, with a median length of 36bp. Data was simulated using multiple read lengths from 150bp to 275bp in 25-bp increments. To verify the synthetic data results, cell lines and reference samples with previously characterized ITDs of varying lengths in the FLT3 gene were sequenced. Libraries were constructed using the Pillar® ONCO/Reveal™ Myeloid Panel and then sequenced on an Illumina® MiSeq® instrument. Secondary analysis was conducted using PiVAT. ITDs with lengths of 21bp, 30bp, 33bp, 42bp, and 126bp were present in the samples and used to evaluate the detection capabilities of PiVAT.

Results:
The simulation data demonstrated that with a read length of 150bp, PiVAT was able to detect ITD lengths up to 81bp, which represented >94% of the 208 ITDs that were in the cohort. Increasing the read length to 175bp allowed further detection of ITDs up to 117bp enabling detection of > 98% of the ITDs present in the study. Analysis of the experimental sequencing data from the real samples demonstrated strong agreement with the synthetic results. PiVAT was able to detect all but the 126-bp ITD at a read length of 150bp, and by increasing the read length to 175bp, PiVAT was able to detect all of the ITDs. This indicated the ability to improve detection capability by increasing read length for even larger ITDs if desired.

Conclusions:
The results demonstrated that PiVAT is capable of reliably detecting ITDs of up to 81bp using a read length of 150bp. In addition, by sequencing at a longer read length of 175 bp or greater, PiVAT is able to detect ITDs of 126bp or longer.