Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Nature Protocols
J QuickN J Loman


Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the ...Continue Reading


Feb 1, 1994·PCR Methods and Applications·S KwokA Wang
Apr 5, 2007·Nature Protocols·Elizabeth Scotto-LavinoMichael A Frohman
Jun 8, 2007·Emerging Infectious Diseases·Steven L SalzbergIlaria Capua
Feb 8, 2008·The New England Journal of Medicine·Gustavo PalaciosW Ian Lipkin
Jun 10, 2009·Bioinformatics·Heng Li1000 Genome Project Data Processing Subgroup
Dec 8, 2009·Bioinformatics·Iain MilneDavid Marshall
Dec 10, 2009·Nature Reviews. Genetics·Michael L Metzker
Jan 30, 2010·Nature Methods·Lira MamanovaDaniel Turner
Jan 12, 2011·Nature Biotechnology·James T RobinsonJill P Mesirov
Apr 18, 2012·Journal of Computational Biology : a Journal of Computational Molecular Cell Biology·Anton BankevichPavel A Pevzner
Apr 24, 2012·Nature Biotechnology·N J LomanMark J Pallen
Aug 22, 2012·Bioinformatics·Johannes Köster, Sven Rahmann
Apr 4, 2014·Bioinformatics·Anthony M BolgerBjoern Usadel
Jun 5, 2014·The New England Journal of Medicine·Michael R WilsonCharles Y Chiu
Aug 27, 2014·Advances in Bioinformatics·Shea N GardnerMonica K Borucki
Aug 16, 2015·Cell·Kristian G AndersenPardis C Sabeti
Jan 26, 2016·Vector Borne and Zoonotic Diseases·Michael J MisencikPhilip M Armstrong
Jan 27, 2016·Emerging Infectious Diseases·Thomas HoenenHeinz Feldmann
Feb 4, 2016·Nature·J QuickMiles Carroll
May 18, 2016·Nature Reviews. Genetics·Sara GoodwinW Richard McCombie
May 19, 2016·Emerging Infectious Diseases·Andy KilianskiC Nicole Rosenzweig
Jan 1, 2015·Genome Biology·Jennifer GardyAndrew Rambaut
Jul 23, 2016·Emerging Infectious Diseases·Samia N NaccacheCharles Y Chiu
Sep 30, 2016·Genome Medicine·Nuno Rodrigues FariaOliver G Pybus
Oct 14, 2016·Nature·E C HolmesKristian G Andersen
Oct 16, 2016·Genome Announcements·Barry AtkinsonRoger Hewson
Jan 17, 2017·Nature Reviews. Microbiology·Charlotte J HouldcroftJudith Breuer
Feb 25, 2017·Genome Announcements·S MárquezChristopher H Logue
May 26, 2017·Nature·Hayden C MetskyPardis C Sabeti


Mar 29, 2018·PLoS Pathogens·Matthew T AliotaDavid H O'Connor
Feb 7, 2018·Frontiers in Microbiology·Raj K SinghRubén Bueno-Marí
Dec 15, 2017·Frontiers in Cellular and Infection Microbiology·Mathilde GondardSara Moutailler
Aug 6, 2018·Applied Microbiology and Biotechnology·Tiphaine C MartinMario Falchi
Aug 25, 2017·Journal of Clinical Microbiology·Richard J N AllcockDavid Warrilow
Oct 13, 2017·Journal of Clinical Microbiology·Jamie K LemonJohn P Dekker
Jul 27, 2018·Annual Review of Virology·Patrick T DolanRaul Andino
Jan 23, 2019·PLoS Computational Biology·David J HawSteven Riley
Mar 9, 2018·Scientific Reports·Philipp KirstahlerSünje Johanna Pamp
Mar 8, 2019·PLoS Neglected Tropical Diseases·Felipe Gomes NavecaNuno Rodrigues Faria
May 26, 2017·Nature·Hayden C MetskyPardis C Sabeti
Apr 18, 2019·PLoS Neglected Tropical Diseases·Anna S JaegerMatthew T Aliota
Mar 29, 2019·Nature Reviews. Genetics·Charles Y Chiu, Steven A Miller
Oct 3, 2019·Journal of Medical Primatology·James CurlinRamesh Akkina
Nov 7, 2018·Scientific Reports·Michael D GallagherDaniel J Macqueen
Nov 12, 2019·The Journal of Infectious Diseases·S PollettN D Grubaugh
Jan 22, 2020·PLoS Neglected Tropical Diseases·Emma L WiseSteven T Pullan
Dec 6, 2019·Journal of Virology·Bernardo GutierrezOliver G Pybus
Oct 24, 2019·Proceedings. Biological Sciences·Jennifer L MalmbergSue VandeWoude
Feb 29, 2020·Infection Control and Hospital Epidemiology : the Official Journal of the Society of Hospital Epidemiologists of America·Erica S ShenoyDavid C Hooper
Mar 6, 2020·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Salman L ButtJames B Stanton
Mar 13, 2020·PloS One·Helder Ricas RezendeCreuza Rachel Vicente
Jun 12, 2019·PloS One·Joilson XavierLuiz Carlos Junior Alcantara
Nov 1, 2019·Journal of Clinical Microbiology·Kuiama LewandowskiPhilippa C Matthews
Oct 11, 2019·Journal of Virology·Marta GiovanettiAna Maria Bispo de Filippis
May 20, 2020·Memórias do Instituto Oswaldo Cruz·Jaqueline Goes de JesusCamila Malta Romano
Apr 30, 2020·Nature·Cheri M AckermanPardis C Sabeti

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