publication . Preprint . Article . Other literature type . 2017

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

Lia Laura Lewis-Ximenez; Jaqueline Goes de Jesus; Marta Giovanetti; Joshua Quick; Glenn Oliveira; Matthew Loose; Jared T. Simpson; Miles W. Carroll; Dennis R. Burton; Ester C. Sabino; ...
Open Access
  • Published: 24 May 2017
  • Publisher: Cold Spring Harbor Laboratory
  • Country: United Kingdom
Abstract
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 co...
Subjects
free text keywords: Article, General Biochemistry, Genetics and Molecular Biology, Illumina dye sequencing, Minion, Genome, Computational biology, Nanopore sequencing, Metagenomics, Multiplex polymerase chain reaction, Biology, DNA sequencing, Zika virus, biology.organism_classification, Computer science
Funded by
NIH| Scripps Translational Science Institute
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 4UL1TR001114-04
  • Funding stream: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
,
NSF| Graduate Research Fellowship Program (GRFP)
Project
  • Funder: National Science Foundation (NSF)
  • Project Code: 1256082
  • Funding stream: Directorate for Education & Human Resources | Division of Graduate Education
,
NIH| Immunology Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5T32AI007244-33
  • Funding stream: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
,
NIH| Understanding Transmission with Integrated Genetic and Epidemiologic Inference
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5U54GM111274-02
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
WT
Project
  • Funder: Wellcome Trust (WT)

1. Gardy, J., Loman, N. J. & Rambaut, A. Real-time digital pathogen surveillance - the time is now.

2. Andersen, K. G. ​et al. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus. ​Cell ​162, 3. Holmes, E. C., Dudas, G., Rambaut, A. & Andersen, K. G. The evolution of Ebola virus: Insights 9. Wilson, M. R. ​et al. Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing.

10. Metzker, M. L. Sequencing technologies-the next generation. ​Nat. Rev. Genet. ​11,​ 31-46 (2010).

11. Loman, N. J. ​et al. Performance comparison of benchtop high-throughput sequencing platforms.

12. Goodwin, S., McPherson, J. D. & McCombie, W. R. Coming of age: ten years of next-generation sequencing technologies. ​Nat. Rev. Genet. ​17,​ 333-351 (2016).

13. Gire, S. K. ​et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 14. Carroll, M. W. ​et al. Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West 15. Faria, N. R. ​et al. Mobile real-time surveillance of Zika virus in Brazil. ​Genome Med. ​8,​ 97 (2016).

16. Matranga, C. B. ​et al. Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA 21. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers.

22. Kwok, S. ​et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. ​Nucleic Acids Res. ​18,​ 999-1005 (1990).

23. Kwok, S., Chang, S. Y., Sninsky, J. J. & Wang, A. A guide to the design and use of mismatched and degenerate primers. ​PCR Methods Appl. ​3,​ S39-47 (1994). [OpenAIRE]

24. Chan, M. ​et al. A Novel System Control for Quality Control of Diagnostic Tests Based on 25. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data.

26. Li, H. ​et al. The Sequence Alignment/Map format and SAMtools. ​Bioinformatics ​25,​ 2078-2079 27. Köster, J. & Rahmann, S. Snakemake-a scalable bioinformatics workflow engine. ​Bioinformatics 29. Robinson, J. T. ​et al. Integrative genomics viewer. ​Nat. Biotechnol. ​29,​ 24-26 (2011).

30. Milne, I. ​et al. Tablet-next generation sequence assembly visualization. ​Bioinformatics ​26, 34. Guerbois, M. ​et al. Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First 36. Misencik, M. J., Grubaugh, N. D., Andreadis, T. G., Ebel, G. D. & Armstrong, P. M. Isolation of a 43. Naccache, S. N. ​et al. Distinct Zika Virus Lineage in Salvador, Bahia, Brazil. ​Emerg. Infect. Dis. ​22, 44. Connor, T. R. ​et al. CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online AUTHOR CONTRIBUTIONS J.Q. and N.J.L conceived the project. J.Q., N.D.G., K.G.A and N.J.L. designed the experiments and wrote the manuscript. J.Q., A.S. and O.G.P. designed the online primer design tool. J.T.S. modified nanopolish to support R9/R9.4 data and indels. M.L. wrote the demultiplexing software. N.J.L. designed and implemented the MinION bioinformatics pipeline. N.D.G., K.G., G.O., R.R-S. and K.G.A. developed the Illumina sequencing protocol and bioinformatics pipeline. L.L.L-X collected the

Abstract
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 co...
Subjects
free text keywords: Article, General Biochemistry, Genetics and Molecular Biology, Illumina dye sequencing, Minion, Genome, Computational biology, Nanopore sequencing, Metagenomics, Multiplex polymerase chain reaction, Biology, DNA sequencing, Zika virus, biology.organism_classification, Computer science
Funded by
NIH| Scripps Translational Science Institute
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 4UL1TR001114-04
  • Funding stream: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
,
NSF| Graduate Research Fellowship Program (GRFP)
Project
  • Funder: National Science Foundation (NSF)
  • Project Code: 1256082
  • Funding stream: Directorate for Education & Human Resources | Division of Graduate Education
,
NIH| Immunology Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5T32AI007244-33
  • Funding stream: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
,
NIH| Understanding Transmission with Integrated Genetic and Epidemiologic Inference
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5U54GM111274-02
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
WT
Project
  • Funder: Wellcome Trust (WT)

1. Gardy, J., Loman, N. J. & Rambaut, A. Real-time digital pathogen surveillance - the time is now.

2. Andersen, K. G. ​et al. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus. ​Cell ​162, 3. Holmes, E. C., Dudas, G., Rambaut, A. & Andersen, K. G. The evolution of Ebola virus: Insights 9. Wilson, M. R. ​et al. Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing.

10. Metzker, M. L. Sequencing technologies-the next generation. ​Nat. Rev. Genet. ​11,​ 31-46 (2010).

11. Loman, N. J. ​et al. Performance comparison of benchtop high-throughput sequencing platforms.

12. Goodwin, S., McPherson, J. D. & McCombie, W. R. Coming of age: ten years of next-generation sequencing technologies. ​Nat. Rev. Genet. ​17,​ 333-351 (2016).

13. Gire, S. K. ​et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 14. Carroll, M. W. ​et al. Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West 15. Faria, N. R. ​et al. Mobile real-time surveillance of Zika virus in Brazil. ​Genome Med. ​8,​ 97 (2016).

16. Matranga, C. B. ​et al. Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA 21. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers.

22. Kwok, S. ​et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. ​Nucleic Acids Res. ​18,​ 999-1005 (1990).

23. Kwok, S., Chang, S. Y., Sninsky, J. J. & Wang, A. A guide to the design and use of mismatched and degenerate primers. ​PCR Methods Appl. ​3,​ S39-47 (1994). [OpenAIRE]

24. Chan, M. ​et al. A Novel System Control for Quality Control of Diagnostic Tests Based on 25. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data.

26. Li, H. ​et al. The Sequence Alignment/Map format and SAMtools. ​Bioinformatics ​25,​ 2078-2079 27. Köster, J. & Rahmann, S. Snakemake-a scalable bioinformatics workflow engine. ​Bioinformatics 29. Robinson, J. T. ​et al. Integrative genomics viewer. ​Nat. Biotechnol. ​29,​ 24-26 (2011).

30. Milne, I. ​et al. Tablet-next generation sequence assembly visualization. ​Bioinformatics ​26, 34. Guerbois, M. ​et al. Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First 36. Misencik, M. J., Grubaugh, N. D., Andreadis, T. G., Ebel, G. D. & Armstrong, P. M. Isolation of a 43. Naccache, S. N. ​et al. Distinct Zika Virus Lineage in Salvador, Bahia, Brazil. ​Emerg. Infect. Dis. ​22, 44. Connor, T. R. ​et al. CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online AUTHOR CONTRIBUTIONS J.Q. and N.J.L conceived the project. J.Q., N.D.G., K.G.A and N.J.L. designed the experiments and wrote the manuscript. J.Q., A.S. and O.G.P. designed the online primer design tool. J.T.S. modified nanopolish to support R9/R9.4 data and indels. M.L. wrote the demultiplexing software. N.J.L. designed and implemented the MinION bioinformatics pipeline. N.D.G., K.G., G.O., R.R-S. and K.G.A. developed the Illumina sequencing protocol and bioinformatics pipeline. L.L.L-X collected the

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