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Updated: Aug 27, 2025

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
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Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory.

Kelly E Craven1, Catherine G Fischer2, LiQun Jiang1

  • 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

The Journal of Molecular Diagnostics : JMD
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Next-generation sequencing (NGS) struggles with detecting insertions and deletions (InDels) due to misaligned reads. The ABRA2 software significantly improves InDel detection and variant allele frequency accuracy in clinical cancer sequencing.

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Area of Science:

  • Genomics
  • Bioinformatics
  • Cancer Research

Background:

  • Short-read next-generation sequencing (NGS) faces challenges in accurately detecting insertions and deletions (InDels).
  • Misaligned reads are common in NGS data, particularly with increasing InDel size, leading to undercalling variant allele frequencies and incorrect variant calls.
  • Existing clinical NGS pipelines may fail to detect or accurately quantify clinically significant variants like fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs).

Purpose of the Study:

  • To systematically analyze the impact of misaligned reads on InDel and FLT3 ITD detection in clinical NGS.
  • To evaluate the efficacy of the Assembly-Based ReAligner (ABRA2) software in improving the detection and quantification of InDels and FLT3 ITDs.
  • To assess ABRA2's performance on various InDel sizes and FLT3 ITDs in clinical malignancy cases.

Main Methods:

  • Analysis of 46 clinical malignancy cases using a clinical NGS assay.
  • Systematic evaluation of short InDels (1-30 bp) and FLT3 ITDs (6-183 bp).
  • Application of Assembly-Based ReAligner (ABRA2) to re-map reads and correct mismapped bases.

Main Results:

  • Misaligned reads were detected in all cases, increasing with InDel size, leading to undercalled variant allele frequencies (1-84%) and incorrect variant calls.
  • ABRA2 corrected 41-100% of mismapped reads, increasing variant allele frequency by 1-61% and correcting most single-base substitution errors.
  • ABRA2 successfully detected multiple FLT3 ITD clones for variants <100 bases, demonstrating its effectiveness for short InDels and smaller FLT3 ITDs.

Conclusions:

  • Misaligned reads pose a significant challenge for accurate InDel and FLT3 ITD detection in clinical NGS.
  • ABRA2 is a valuable tool for improving the accuracy of InDel and FLT3 ITD detection and quantification in clinical sequencing.
  • ABRA2 demonstrates robust performance for short InDels and FLT3 ITDs under 100 bases, enhancing diagnostic capabilities in oncology.