Advertisement

Multiplexed Targeted Sequencing for Oxford Nanopore MinION: A Detailed Library Preparation Procedure

  • Timokratis KaramitrosEmail author
  • Gkikas MagiorkinisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1712)

Abstract

MinION is a small form factor sequencer recently retailed by Oxford Nanopore technologies. This lighter-sized USB3.0-interfaced device uses innovative nanotechnology to generate extra-long reads from libraries prepared using only standard molecular biology lab equipment. The flexibility and the portability of the platform makes it ideal for point-of-interest and real-time surveillance applications. However, MinION’s limited capacity is not enough for the study of specific targets within larger genomes. Apart from just PCR-amplifying regions of interest, the capture of long reads spanning the edges of known-unknown genomic regions is of great importance for structural studies, such as the identification of mobile elements’ integrations sites, bridging over low complexity repetitive regions etc.

In this study, using MinION-kit-included and commercially available reagents, we have developed an easy and versatile wet-lab procedure for the targeted enrichment of MinION libraries, capturing DNA fragments of interest before the ligation of the sensitive MinION sequencing-adapters. This method allows for simultaneous target-enrichment and barcode-multiplexing of up to 12 libraries, which can be loaded in the same sequencing run.

Key words

MinION Target-enrichment Library preparation Baits Hybridization 

References

  1. 1.
    Kasianowicz JJ, Brandin E, Branton D, Deamer DW (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A 93(24):13770–13773CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Branton D, Deamer DW, Marziali A, Bayley H, Benner SA, Butler T, Di Ventra M, Garaj S, Hibbs A, Huang X, Jovanovich SB, Krstic PS, Lindsay S, Ling XS, Mastrangelo CH, Meller A, Oliver JS, Pershin YV, Ramsey JM, Riehn R, Soni GV, Tabard-Cossa V, Wanunu M, Wiggin M, Schloss JA (2008) The potential and challenges of nanopore sequencing. Nat Biotech 26(10):1146–1153. https://doi.org/10.1038/nbt.1495 CrossRefGoogle Scholar
  3. 3.
    Stoddart D, Heron AJ, Mikhailova E, Maglia G, Bayley H (2009) Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc Natl Acad Sci U S A 106(19):7702–7707. https://doi.org/10.1073/pnas.0901054106 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Cherf GM, Lieberman KR, Rashid H, Lam CE, Karplus K, Akeson M (2012) Automated forward and reverse ratcheting of DNA in a nanopore at 5-a precision. Nat Biotech 30(4):344–348. https://doi.org/10.1038/nbt.2147 CrossRefGoogle Scholar
  5. 5.
    Watson M, Thomson M, Risse J, Talbot R, Santoyo-Lopez J, Gharbi K, Blaxter M (2014) poRe: an R package for the visualization and analysis of nanopore sequencing data. Bioinformatics 31(1):114–115. https://doi.org/10.1093/bioinformatics/btu590 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Jain M, Fiddes IT, Miga KH, Olsen HE, Paten B, Akeson M (2015) Improved data analysis for the MinION nanopore sequencer. Nat Meth 12(4):351–356. https://doi.org/10.1038/nmeth.3290 CrossRefGoogle Scholar
  7. 7.
    Hodges E, Xuan Z, Balija V, Kramer M, Molla MN, Smith SW, Middle CM, Rodesch MJ, Albert TJ, Hannon GJ, McCombie WR (2007) Genome-wide in situ exon capture for selective resequencing. Nat Genet 39(12):1522–1527. https://doi.org/10.1038/ng.2007.42 CrossRefPubMedGoogle Scholar
  8. 8.
    Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotech 27(2):182–189. https://doi.org/10.1038/nbt.1523 CrossRefGoogle Scholar
  9. 9.
    Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ (2010) Target-enrichment strategies for next-generation sequencing. Nat Meth 7(2):111–118. https://doi.org/10.1038/nmeth.1419 CrossRefGoogle Scholar
  10. 10.
    Briggs AW, Good JM, Green RE, Krause J, Maricic T, Stenzel U, Lalueza-Fox C, Rudan P, Brajkovic D, Kucan Z, Gusic I, Schmitz R, Doronichev VB, Golovanova LV, de la Rasilla M, Fortea J, Rosas A, Paabo S (2009) Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325(5938):318–321. https://doi.org/10.1126/science.1174462 CrossRefPubMedGoogle Scholar
  11. 11.
    Maricic T, Whitten M, Paabo S (2010) Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. PLoS One 5(11):e14004. https://doi.org/10.1371/journal.pone.0014004 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Karamitros T, Magiorkinis G (2015) A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits. Nucl Acid Res 43(22):e152. https://doi.org/10.1093/nar/gkv773 CrossRefGoogle Scholar
  13. 13.
    David M, Dursi LJ, Yao D, Boutros PC, Simpson JT (2016) Nanocall: an open source basecaller for oxford nanopore sequencing data. bioRxiv 33(1). https://doi.org/10.1101/046086
  14. 14.
    Frith MC, Hamada M, Horton P (2010) Parameters for accurate genome alignment. BMC Bioinform 11:80. https://doi.org/10.1186/1471-2105-11-80 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of ZoologyUniversity of OxfordOxfordUK
  2. 2.Department of Medical MicrobiologyHellenic Pasteur InstituteAthensGreece

Personalised recommendations