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Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis., (© 2022. The Author(s).)

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort ( n = 37) including patients with various neurogenetic diseases ( n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.