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

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

Authors :
Coope RJN
Matic N
Pandoh PK
Corbett RD
Smailus DE
Pleasance S
Lowe CF
Ritchie G
Chorlton SD
Young M
Ally AA
Asano JK
Carlsen RE
Chahal SS
Zhao Y
Holmes DT
Romney MG
Jones SJM
Marra MA
Source :
The journal of applied laboratory medicine [J Appl Lab Med] 2022 Sep 01; Vol. 7 (5), pp. 1025-1036.
Publication Year :
2022

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.<br />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.<br />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.<br />Conclusions: We demonstrated a robust automated sequencing pipeline that takes advantage of input Ct values to improve reliability.<br />Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: S.D. Chorlton, PathoGene Inc., BugSeq Bioinformatics Inc. Honoraria: None declared. Research Funding: Researchers from the St. Paul’s Hospital Department of Pathology and Laboratory Medicine have received institutional support from the Public Health Agency of Canada’s COVID-19 Immunity Task Force Hot Spots Competitive Grant, 2021-HQ-000120 (M.G. Romney, N. Matic, G. Ritchie, C.F. Lowe, M. Young), Genome BC COVID-19 rapid response grant (M.G. Romney, N. Matic, G. Ritchie, C.F. Lowe, M. Young), Genome BC Rapid SARS-CoV-2 Vaccine Research Initiative in BC (M.G. Romney and C.F. Lowe), Providence Health Care Research Institute (N. Matic), and Canada Foundation for Innovation Exceptional Opportunities Fund–COVID-19 (C.F. Lowe). M.A. Marra, S. Jones, D.E. Smailus, R. Corbett, S. Pleasance, S. Chahal, R.E. Carlsen, R.J.N. Coope, P.K. Pandoh, J. Asano, A. Ally, Y. Zhao, and Canada’s Michael Smith Genome Sciences Centre, have also received institutional support from Genome BC (COV-088), Genome Canada (262SEQ), and the Canada Foundation for Innovation and BC Knowledge Development Fund (#40991). S.D. Chorlton has received funding from the Open Philanthropy Project. Expert Testimony: None declared. Patents: None declared. Other Remuneration: D. Holmes has received travel reimbursements from AACC and MSACL.<br /> (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Details

Language :
English
ISSN :
2576-9456
Volume :
7
Issue :
5
Database :
MEDLINE
Journal :
The journal of applied laboratory medicine
Publication Type :
Academic Journal
Accession number :
35723286
Full Text :
https://doi.org/10.1093/jalm/jfac054