Sequencing by binding rivals SMOR error-corrected sequencing by synthesis technology for accurate detection and quantification of minor (< 0.1%) subpopulation variants

Abstract

Background: Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications, including the detection of drug resistant pathogens and somatic variant detection in oncology. A recently available sequencing approach termed 'sequencing by binding (SBB)' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using SBB for the detection of ultra-rare drug resistant subpopulations in Mycobacterium tuberculosis (Mtb) using a targeted amplicon assay and compares the performance of SBB to single molecule overlapping reads (SMOR) error corrected sequencing by synthesis (SBS) data.

Results: SBS displayed an elevated error rate when compared to SMOR error-corrected SBS and SBB techniques. SMOR error-corrected SBS and SBB technologies performed similarly within the linear range studies and error rate studies.

Conclusions: With lower sequencing error rates within SBB sequencing, this technique looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.

Keywords: Heteroresistance; NGS benchmarking; Sequencing by binding (SBB); Sequencing by synthesis (SBS); Single molecule overlapping reads (SMOR); Tuberculosis (TB); Ultra-rare mutation detection.

Fig. 1

Alignment of SBS, SBS-SMOR, and SBB reads for katG (A) and gyrA (B) Cartoon at the top of each IGV plot shows the relative position of the sequencing reads where SBS had both read 1 and read 2 each at 300 bps, while SBB has only read 1 from the 3’ end with variable lengths up to 150 bps long. Images in IGV are representatives of the the 10% proportion mixture and each read is colored grey, in squished mode, and are grouped by katG PCR position 224 (gene mutation: g944c) or gryA PCR position 197 (gene mutation: a241g). Mutations compared to the plasmid reference include single nucleotide variants, A(Green), G (Orange), C (Blue), T (Red), and N (Dark Grey)

Fig. 2

Average observed % error rate (log 10) across gyrA and katG genes for comparable positions. The line represents the mean total error rate across samples at the specified position with a 95% confidence interval displayed as the lighter-colored ribbon