Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Abstract

Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

References 12

1. 1.

   Manning JE Bohl JA Lay S et al Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia. bioRxiv 2020:2020.03.02.968818.

   Not in our library

2. 2.

   Gohl DM Garbe J Grady P et al A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2. bioRxiv2020, doi: 10.1101/2020.05.11.088724.

   Not in our library10.1186/s12864-020-07283-6

3. 3.

   Moore SC Penrice-Randal R Alruwaili M et al Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19. medRxiv2020, doi: 10.1101/2020.03.05.20032011.

   Not in our library10.3390/v12101164

4. 4.

   Itokawa K Sekizuka T Hashino M et al A proposal of alternative primers for the ARTIC Network’s multiplex PCR to improve coverage of SARS-CoV-2 genome sequencing. BioRxiv2020, doi: 10.1101/2020.03.10.985150.

   Not in our library

5. 5.

   Resende PC Motta FC Roy S et al SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms. bioRxiv2020, doi: 10.1101/2020.04.30.069039.

   Not in our library

6. 6.

   Eden JS Rockett R Carter I et al; 2019-nCoV Study Group. An emergent clade of SARS-CoV-2 linked to returned travellers from Iran. Virus Evol2020;6:veaa027.

   Not in our library10.1093/ve/veaa027

7. 7.
   Data, disease and diplomacy: GISAID's innovative contribution to global health · 2017

   No notice
8. 8.

   Artic-ncov. 2019 https://github.com/artic-network/artic-ncov2019 (1 May 2020, date last accessed).

   Not in our library

9. 9.

   Seqtk LH. https://github.com/lh3/seqtk (5 February 2014, date last accessed).

   Not in our library

10. 10.

    Quick J Grubaugh ND Pullan ST et al Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc2017;12:1261–76.

    Not in our library10.1038/nprot.2017.066

11. 11.

    Quick J. nCoV-2019 sequencing protocol v2 v1 (protocols.io.bdp7i5rn). protocols.io.

    Not in our library

12. 12.

    Wang W Xu Y Gao R et al Detection of SARS-CoV-2 in different types of clinical specimens. JAMA2020;323:1843–4.

    Not in our library10.1001/jama.2020.3786

Cited by papers we hold 5

• SARS-CoV-2 3CL pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376 · 2023
  No notice
• The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance · 2022
  No notice
• Exploiting genomic surveillance to map the spatio-temporal dispersal of SARS-CoV-2 spike mutations in Belgium across 2020 · 2021
  Not checked
• Emergence and expansion of SARS-CoV-2 B.1.526 after identification in New York · 2021
  No notice
• Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation · 2020
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.