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Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR

View ORCID ProfileKentaro Itokawa, View ORCID ProfileTsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, View ORCID ProfileMakoto Kuroda
doi: https://doi.org/10.1101/2020.03.10.985150
Kentaro Itokawa
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Toyama 1-23-1, Shinjuku-ku, Tokyo, Japan
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  • For correspondence: itokawa@nih.go.jp
Tsuyoshi Sekizuka
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Toyama 1-23-1, Shinjuku-ku, Tokyo, Japan
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Masanori Hashino
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Toyama 1-23-1, Shinjuku-ku, Tokyo, Japan
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Rina Tanaka
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Toyama 1-23-1, Shinjuku-ku, Tokyo, Japan
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Makoto Kuroda
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Toyama 1-23-1, Shinjuku-ku, Tokyo, Japan
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Abstract

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample’s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Several modifications for methods and new results were added.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 01, 2020.
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Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR
Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
bioRxiv 2020.03.10.985150; doi: https://doi.org/10.1101/2020.03.10.985150
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Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR
Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
bioRxiv 2020.03.10.985150; doi: https://doi.org/10.1101/2020.03.10.985150

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