Sequencing by avidity enables high accuracy with low reagent consumption

Abstract

We present avidity sequencing - a novel sequencing chemistry that separately optimizes the process of stepping along a DNA template and the process of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar, and yield negligible dissociation rates. We demonstrate the use of avidites as a key component of a sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing. We also show the advantages of this technology in sequencing through long homopolymers.

Characterizing and addressing error modes to improve sequencing accuracy

“…Independent optimization of the stepping and resolving processes with respect to reagent recipes and fit-for-purpose enzymes is an important contributor to ABC accuracy. We demonstrated that accuracy via quality score recalibration of sequencing data, which showed that the quality score assignments above Q40 agreed with empirically determined accuracy 7 . Base quality score recalibration (BQSR) works by considering the assigned base calls and quality scores (along with several covariates) and true calls determined via read alignment to a well-characterized reference genome 8 .…”

Low-pass sequencing plus imputation using avidity sequencing displays comparable imputation accuracy to sequencing by synthesis while reducing duplicates

“…For the last decade, sequencing-by-synthesis has been the predominant method underlying high-throughput short-read whole-genome-sequencing. Recently, a number of alternative methods such as avidity sequencing and sequencing-by-binding have been proposed for highly parallel high-throughput sequencing (Arslan et al, 2022; Pacific Biosciences, 2022). In this study, we examined the viability of avidity sequencing as an alternative to SBS in the context of low-pass whole genome sequencing followed by imputation.…”

“…Avidity sequencing is described in Arslan et al (2022). Briefly, linear library molecules are circularized through hybridization to splint oligos followed by ligation.…”

An efficient design for whole genome trio sequencing identifies key variants in rare neurological disorder cases

“…We chose to sequence the proband at a typical coverage level of approximately 35-40X and the parents at half that coverage (∼15-20X). The design is efficient in cost and time because the three genomes can be sequenced in a single flowcell of the AVITI 6 benchtop sequencing instrument. We hypothesized that we would be able to observe inheritance patterns and identify de novo variants even with the reduced coverage in the parents.…”