Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Abstract

Detection of insertions and deletions (InDels) by short-read next-generation sequencing (NGS) technology can be challenging because of frequent misaligned reads. A systematic analysis of short InDels (1 to 30 bases) and fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs; 6 to 183 bases) from 46 clinical cases of solid or hematologic malignancy processed with a clinical NGS assay identified misaligned reads in every case, ranging from 3% to 100% of reads with the InDel showing mismapped bases. Mismaps also increased with InDel size. As a consequence, the clinical NGS bioinformatics pipeline undercalled the variant allele frequency by 1% to 84%, incorrectly called simultaneous single-base substitutions along with InDels, or did not report an FLT3 ITD that had been detected by capillary electrophoresis. To improve the ability of the pipeline to better detect and quantify InDels, we utilized a software program called Assembly-Based ReAligner (ABRA2) to more accurately remap reads. ABRA2 was able to correct 41% to 100% of the reads with mismapped bases and led to absolute increases in the variant allele frequency from 1% to 61% along with correction of all of the single-base substitutions except for two cases. ABRA2 could also detect multiple FLT3 ITD clones except for one 183-base ITD. Our analysis has found that ABRA2 performs well on short InDels as well as FLT3 ITDs that are <100 bases.