Skip to main content

Reduced Representation Methods for Subgenomic Enrichment and Next-Generation Sequencing

  • Protocol
  • First Online:
Molecular Methods for Evolutionary Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 772))

Abstract

Several methods have been developed to enrich DNA for subsets of the genome prior to next-generation sequencing. These front-end enrichment strategies provide powerful and cost-effective tools for researchers interested in collecting large-scale genomic sequence data. In this review, I provide an overview of both general and targeted reduced representation enrichment strategies that are commonly used in tandem with next-generation sequencing. I focus on several key issues that are likely to be important when deciding which enrichment strategy is most appropriate for a given experiment. Overall, these techniques can enable the collection of large-scale genomic data in diverse species, providing a powerful tool for the study of evolutionary biology.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Mamanova L, Coffey AJ, Scott CE et al (2010) Target-enrichment strategies for next-generation sequencing. Nat Methods 7:111–118

    Article  PubMed  CAS  Google Scholar 

  2. Turner EH, Ng SB, Nickerson DA et al (2009) Methods for genomic partitioning. Ann Rev Genomics Hum Genet 10:263–284

    Article  CAS  Google Scholar 

  3. Lee H, O’Connor BD, Merriman B et al (2009) Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. BMC Genomics 10:646

    Article  PubMed  Google Scholar 

  4. Summerer D (2009) Enabling technologies of genomic-scale sequence enrichment for targeted high-throughput sequencing. Genomics 94:363–368

    Article  PubMed  CAS  Google Scholar 

  5. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  PubMed  CAS  Google Scholar 

  6. Metzker ML (2010) Sequencing technologies - the next generation. Nat Rev Genet 11:31–46

    Article  PubMed  CAS  Google Scholar 

  7. Shendure J, Ji HL (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145

    Article  PubMed  CAS  Google Scholar 

  8. Margulies M, Egholm M, Altman WE et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380

    PubMed  CAS  Google Scholar 

  9. Bentley DR, Balasubramanian S, Swerdlow HP et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59

    Article  PubMed  CAS  Google Scholar 

  10. Valouev A, Ichikawa J, Tonthat T et al (2008) A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Res 18:1051–1063

    Article  PubMed  CAS  Google Scholar 

  11. Wheeler DA, Srinivasan M, Egholm M et al (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452:872–876

    Article  PubMed  CAS  Google Scholar 

  12. Wang J, Wang W, Li R et al (2008) The diploid genome sequence of an Asian individual. Nature 456:60–U61

    Article  PubMed  CAS  Google Scholar 

  13. Li RQ, Fan W, Tian G et al (2010) The sequence and de novo assembly of the giant panda genome. Nature 463:311–317

    Article  PubMed  CAS  Google Scholar 

  14. Altshuler D, Pollara VJ, Cowles CR et al (2000) An SNP map of the human genome generated by reduced representation shotgun sequencing. Nature 407:513–516

    Article  PubMed  CAS  Google Scholar 

  15. Van Tassell CP, Smith TP, Matukamalli LK et al (2008) SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat Methods 5:247–252

    Article  PubMed  Google Scholar 

  16. Baird NA, Etter PD, Atwood TS et al (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3:e3376

    Article  PubMed  Google Scholar 

  17. Wiedmann RT, Smith TPL, Nonneman DJ (2008) SNP discovery in swine by reduced representation and high throughput pyrosequencing. BMC Genet 9:81

    Article  PubMed  Google Scholar 

  18. Hohenlohe PA, Bassham S, Etter PD et al (2010) Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genet. 6:e1000862

    Article  PubMed  Google Scholar 

  19. Van Bers NEM, Van Oers K, Kerstens HHD et al (2010) Genome-wide SNP detection in the great tit Parus major using high throughput sequencing. Mol Ecol 19:89–99

    Article  PubMed  Google Scholar 

  20. Adams MD, Kelley JM, Gocayne JD et al (1991) Complementary DNA sequencing: Expressed sequence tags and the human genome project. Science 252:1651–1656

    Article  PubMed  CAS  Google Scholar 

  21. Velculescu VE, Zhang L, Vogelstein B et al (1995) Serial analysis of gene expression. Science 270:484–487

    Article  PubMed  CAS  Google Scholar 

  22. Velculescu VE, Zhang L, Zhou W et al (1997) Characterization of the yeast transcriptome. Cell 88:243–251

    Article  PubMed  CAS  Google Scholar 

  23. Mortazavi A, Williams BA, McCue K et al (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628

    Article  PubMed  CAS  Google Scholar 

  24. Zerbino DR, Birney E (2008) Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829

    Article  PubMed  CAS  Google Scholar 

  25. Su AI, Cooke MP, Ching KA et al (2002) Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci USA 99:4465–4470

    Article  PubMed  CAS  Google Scholar 

  26. Montgomery SB, Sammeth M, Gutierrez-Arcelus M et al (2010) Transcriptome genetics using second generation sequencing in a Caucasian population. Nature 464:773–777

    Article  PubMed  CAS  Google Scholar 

  27. Carninci P, Shibata Y, Hayatsu N et al (2000) Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res 10:1617–1630

    Article  PubMed  CAS  Google Scholar 

  28. Varley KE, Mitra RD (2008) Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Genome Res 18:1844–1850

    Article  PubMed  CAS  Google Scholar 

  29. Stiller M, Knapp M, Stenzel U et al (2009) Direct multiplex sequencing (DMPS): a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA. Genome Res 19:1843–1848

    Article  PubMed  CAS  Google Scholar 

  30. Tewhey R, Warner JB, Nakano M et al (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27:1025–1031

    Article  PubMed  CAS  Google Scholar 

  31. Lovett M, Kere J, Hinton LM (1991) Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc Natl Acad Sci USA 88:9628–9632

    Article  PubMed  CAS  Google Scholar 

  32. Hodges E, Xuan Z, Balija V et al (2007) Genome-wide in situ exon capture for selective resequencing. Nat Genet 39:1522–1527

    Article  PubMed  CAS  Google Scholar 

  33. Albert TJ, Molla MN, Muzny DM et al (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4:903–905

    Article  PubMed  CAS  Google Scholar 

  34. Okou DT, Steinberg KM, Middle C et al (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4:907–909

    Article  PubMed  CAS  Google Scholar 

  35. Gnirke A, Melnikov A, Maguire J et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27:182–189

    Article  PubMed  CAS  Google Scholar 

  36. Cleary MA, Kilian K, Wang Y et al (2004) Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1:241–248

    Article  PubMed  CAS  Google Scholar 

  37. Hughes TR, Mao M, Jones AR et al (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19:342–347

    Article  PubMed  CAS  Google Scholar 

  38. https://earray.chem.agilent.com/earray/

  39. Ng SB, Turner EH, Robertson PD et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276

    Article  PubMed  CAS  Google Scholar 

  40. Ng SB, Buckingham KJ, Lee C et al (2010) Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 42:30–35

    Article  PubMed  CAS  Google Scholar 

  41. Hodges E, Rooks M, Xuan Z et al (2009) Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat Protocols 4:960–974

    Article  CAS  Google Scholar 

  42. Porreca GJ, Zhang K, Li JB et al (2007) Multiplex amplification of large sets of human exons. Nat Methods 4:931–936

    Article  PubMed  CAS  Google Scholar 

  43. Briggs AW, Good JM, Green RE et al (2009) Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325:318–321

    Article  PubMed  CAS  Google Scholar 

  44. Krause J, Fu Q, Good JM et al (2010) The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature 464:894–897

    Article  PubMed  CAS  Google Scholar 

  45. Krause J, Briggs AW, Kircher M et al (2010) A complete mtDNA genome of an early modern human from Kostenki, Russia. Curr Biol 20:231–236

    Article  PubMed  CAS  Google Scholar 

  46. Maricic T, Whitten M, Pääbo S (2010) Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. PLoS ONE 5:e14004

    Article  PubMed  Google Scholar 

  47. Wolf JBW, Bayer T, Haubold B et al (2010) Nucleotide divergence vs. gene expression differentiation: comparative transcriptome sequencing in natural isolates from the carrion crow and its hybrid zone with the hooded crow. Mol Ecol 19:162–175

    Article  PubMed  CAS  Google Scholar 

  48. Burbano HA, Hodges E, Green RE et al (2010) Targeted investigation of the Neandertal genome by array-based sequence capture. Science 328:723–725

    Article  PubMed  CAS  Google Scholar 

  49. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  PubMed  CAS  Google Scholar 

  50. Li H, Handsaker B, Wysoker A et al (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  Google Scholar 

  51. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18:1851–1858

    Article  PubMed  CAS  Google Scholar 

  52. Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10:R25

    Article  PubMed  Google Scholar 

  53. Alkan C, Kidd JM, Marques-Bonet T et al (2009) Personalized copy number and segmental duplication maps using next-generation sequencing. Nat Genet 41:1061–1067

    Article  PubMed  CAS  Google Scholar 

  54. Meyer M, Kircher M (2010) Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb Protoc. doi:10.1101/pdb.prot5448

  55. Blow MJ, Zhang T, Woyke T et al (2008) Identification of ancient remains through geno­mic sequencing. Genome Res 18:1347–1353

    Article  PubMed  CAS  Google Scholar 

  56. Meyer M, Stenzel U, Hofreiter M (2008) Parallel tagged sequencing on the 454 platform. Nature Protocols 3:267–278

    Article  PubMed  CAS  Google Scholar 

  57. Briggs AW, Stenzel U, Johnson PLF et al (2007) Patterns of damage in genomic DNA sequences from a Neandertal. Proc Natl Acad Sci USA 104:14616–14621

    Article  PubMed  CAS  Google Scholar 

  58. Green RE, Krause J, Briggs AW et al (2010) A draft sequence of the Neandertal genome. Science 328:710–722

    Article  PubMed  CAS  Google Scholar 

  59. Green RE, Krause J, Ptak SE et al (2006) Analysis of one million base pairs of Neanderthal DNA. Nature 444:330–336

    Article  PubMed  CAS  Google Scholar 

  60. Meyerhans A, Vartanian JP, Wainhobson S (1990) DNA recombination during PCR. Nucleic Acids Res 18:1687–1691

    Article  PubMed  CAS  Google Scholar 

  61. Botstein D, White RL, Skolnick M et al (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

I thank Emily Hodges, Frank Albert, Martin Kircher, Adrian Briggs, Hernán Burbano, Gordon Luikart, and Matthias Meyer for many helpful conversations on NGS and targeted enrichment. Research contributing to this review was supported by an NSF international postdoctoral fellowship (OISE-0754461).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jeffrey M. Good .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Good, J.M. (2012). Reduced Representation Methods for Subgenomic Enrichment and Next-Generation Sequencing. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-228-1_5

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-227-4

  • Online ISBN: 978-1-61779-228-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics