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Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

View ORCID ProfileElizabeth M. Batty, View ORCID ProfileTheerarat Kochakarn, Arporn Wangwiwatsin, Khajohn Joonlasak, Angkana T. Huang, Bhakbhoom Panthan, Poramate Jiaranai, View ORCID ProfileKrittikorn Kümpornsin, Namfon Kotanan, View ORCID ProfileWudtichai Manasatienkij, Treewat Watthanachockchai, Kingkan Rakmanee, Anthony R. Jones, Stefan Fernandez, Insee Sensorn, Somnuek Sungkanuparph, Ekawat Pasomsub, Chonticha Klungthong, View ORCID ProfileThanat Chookajorn, Wasun Chantratita
doi: https://doi.org/10.1101/2020.06.16.154286
Elizabeth M. Batty
1Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
2Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
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  • For correspondence: elizabeth.b@tropmedres.ac
Theerarat Kochakarn
3Genomics and Evolutionary Medicine Unit (GEM), Center of Excellence in Malaria Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
4Faculty of Medicine Ramathibodi Hospital, Mahidol University Bangkok, Thailand
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Arporn Wangwiwatsin
5Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand
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Khajohn Joonlasak
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Angkana T. Huang
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Bhakbhoom Panthan
7Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Poramate Jiaranai
7Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Krittikorn Kümpornsin
8Wellcome Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK
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Namfon Kotanan
3Genomics and Evolutionary Medicine Unit (GEM), Center of Excellence in Malaria Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
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Wudtichai Manasatienkij
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Treewat Watthanachockchai
10Division of Virology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Kingkan Rakmanee
10Division of Virology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Anthony R. Jones
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Stefan Fernandez
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Insee Sensorn
7Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Somnuek Sungkanuparph
9Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samut Prakan, Thailand
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Ekawat Pasomsub
10Division of Virology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Chonticha Klungthong
6Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
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Thanat Chookajorn
3Genomics and Evolutionary Medicine Unit (GEM), Center of Excellence in Malaria Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
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Wasun Chantratita
7Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Abstract

Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://www.ebi.ac.uk/ena/browser/view/PRJEB38369

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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Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform
Elizabeth M. Batty, Theerarat Kochakarn, Arporn Wangwiwatsin, Khajohn Joonlasak, Angkana T. Huang, Bhakbhoom Panthan, Poramate Jiaranai, Krittikorn Kümpornsin, Namfon Kotanan, Wudtichai Manasatienkij, Treewat Watthanachockchai, Kingkan Rakmanee, Anthony R. Jones, Stefan Fernandez, Insee Sensorn, Somnuek Sungkanuparph, Ekawat Pasomsub, Chonticha Klungthong, Thanat Chookajorn, Wasun Chantratita
bioRxiv 2020.06.16.154286; doi: https://doi.org/10.1101/2020.06.16.154286
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Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform
Elizabeth M. Batty, Theerarat Kochakarn, Arporn Wangwiwatsin, Khajohn Joonlasak, Angkana T. Huang, Bhakbhoom Panthan, Poramate Jiaranai, Krittikorn Kümpornsin, Namfon Kotanan, Wudtichai Manasatienkij, Treewat Watthanachockchai, Kingkan Rakmanee, Anthony R. Jones, Stefan Fernandez, Insee Sensorn, Somnuek Sungkanuparph, Ekawat Pasomsub, Chonticha Klungthong, Thanat Chookajorn, Wasun Chantratita
bioRxiv 2020.06.16.154286; doi: https://doi.org/10.1101/2020.06.16.154286

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