Dataset Information

Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

ABSTRACT:

Background

To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow.

Methods

We used the established ARTIC protocol with approximately 400 bp amplicons sequenced on Oxford Nanopore's MinION. Sequences were analyzed using Nextclade, assigning both a clade and quality score to each sample.

Results

A total of 2179 samples on twenty-five 96-well plates were sequenced. Plates of purified RNA were processed within 12 h, sequencing required up to 24 h, and analysis of each pooled plate required 1 h. The use of samples with known threshold cycle (Ct) values enabled normalization, acted as a quality control check, and revealed a strong correlation between sample Ct values and successful analysis, with 85% of samples with Ct < 30 achieving a "good" Nextclade score. Less abundant samples responded to enrichment with the fraction of Ct > 30 samples achieving a "good" classification rising by 60% after addition of a post-ARTIC PCR normalization. Serial dilutions of 3 variant of concern samples, diluted from approximately Ct = 16 to approximately Ct = 50, demonstrated successful sequencing to Ct = 37. The sample set contained a median of 24 mutations per sample and a total of 1281 unique mutations with reduced sequence read coverage noted in some regions of some samples. A total of 10 separate strains were observed in the sample set, including 3 variants of concern prevalent in British Columbia in the spring of 2021.

Conclusions

We demonstrated a robust automated sequencing pipeline that takes advantage of input Ct values to improve reliability.

SUBMITTER: Coope RJN

PROVIDER: S-EPMC9384306 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

Coope Robin J N RJN Matic Nancy N Pandoh Pawan K PK Corbett Richard D RD Smailus Duane E DE Pleasance Stephen S Lowe Christopher F CF Ritchie Gordon G Chorlton Samuel D SD Young Matthew M Ally Adrian A AA Asano Jennifer K JK Carlsen Rebecca E RE Chahal Sundeep S SS Zhao Yongjun Y Holmes Daniel T DT Romney Marc G MG Jones Steven J M SJM Marra Marco A MA

The journal of applied laboratory medicine 20220901 5

<h4>Background</h4>To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow.<h4>Methods</h4>We used the established ARTIC protocol with approximately 400 bp amplicons sequenced on Oxford Nanopore's MinION. Sequences were analyzed using Nextclade, assigning both a clade and quality score to each sample.<h4>Results</h4>A total of 2179 samples on twenty-five ...[more]

PMID: 35723286

Similar Datasets

Project description:We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Dataset Information

Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

Background

Methods

Results

Conclusions

Publications

Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets