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- Title
Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2<sup> </sup>.
- Authors
Coope, Robin J N; Matic, Nancy; Pandoh, Pawan K; Corbett, Richard D; Smailus, Duane E; Pleasance, Stephen; Lowe, Christopher F; Ritchie, Gordon; Chorlton, Samuel D; Young, Matthew; Ally, Adrian A; Asano, Jennifer K; Carlsen, Rebecca E; Chahal, Sundeep S; Zhao, Yongjun; Holmes, Daniel T; Romney, Marc G; Jones, Steven J M; Marra, Marco A
- 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.
- Subjects
BRITISH Columbia; QUALITY control; SEQUENCE analysis; PATHOLOGICAL laboratories; SARS-CoV-2; OCHRATOXINS
- Publication
Journal of Applied Laboratory Medicine, 2022, Vol 7, Issue 5, p1025
- ISSN
2475-7241
- Publication type
Article
- DOI
10.1093/jalm/jfac054