Dataset Information

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

ABSTRACT: 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.

SUBMITTER: Gunter HM

PROVIDER: S-EPMC9616880 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Gunter Helen M HM Youlten Scott E SE Madala Bindu Swapna BS Reis Andre L M ALM Stevanovski Igor I Wong Ted T Kummerfield Sarah K SK Deveson Ira W IW Santini Nadia S NS Marcellin Esteban E Mercer Tim R TR

Nature communications 20221028 1

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 informati ...[more]

PMID: 36307482

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Project description:BackgroundLong-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT's improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads.ResultsBasecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy's plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific).ConclusionsThe use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.

Dataset Information

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Publications

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

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