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Titration-free 454 sequencing using Y adapters


We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe–based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment. A single quality control step prior to emulsion PCR ensures that libraries contain no adapter dimers and have an optimal length distribution. The presented protocol takes 7 h to prepare eight barcoded libraries from genomic DNA into libraries that are ready to use for full-scale emPCR. It will be useful, for example, to allow analyses of precious clinical samples and amplification-free metatranscriptomics.

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Figure 1: A schematic illustration of the constructions of two types of libraries, A-B and Y.
Figure 2: Design of Y MID adapter.
Figure 3: Quality control.
Figure 4: Anticipated results.


  1. Roche Diagnostics. Rapid Library Preparation Method Manual: GS FLX Titanium Series (Roche Applied Science, October 2009 (Rev. January 2010)).

  2. Frias-Lopez, J. et al. Microbial community gene expression in ocean surface waters. Proc. Natl. Acad. Sci. USA 105, 3805–3810 (2008).

    Article  CAS  Google Scholar 

  3. Aird, D. et al. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 12, R18 (2011).

    Article  CAS  Google Scholar 

  4. Davey, J.L. & Blaxter, M.W. RADSeq: next-generation population genetics. Brief Funct. Genomics 9, 416–423 (2010).

    Article  CAS  Google Scholar 

  5. Zheng, Z. et al. Titration-free massively parallel pyrosequencing using trace amounts of starting material. Nucleic Acids Res. 38, e137 (2010).

    Article  Google Scholar 

  6. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  Google Scholar 

  7. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    Article  CAS  Google Scholar 

  8. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    Article  CAS  Google Scholar 

  9. Baird, N.A. et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3, e3376 (2008).

    Article  Google Scholar 

  10. Carson, S. et al. DNA sequencing by capillary electrophoresis: use of a two-laser-two-window intensified diode array detection system. Anal. Chem. 65, 3219–3226 (1993).

    Article  CAS  Google Scholar 

  11. White, R.A. III, Blainey, P.C., Fan, H.C. & Quake, S.R. Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics 10, 116 (2009).

    Article  Google Scholar 

  12. Huang, J., Zheng, Z., Andersen, A.F., Engstran, L. & Ye, W. Rapid screening of complex DNA samples by single-molecule amplification and sequencing. PLoS ONE 6, e19723 (2011).

    Article  CAS  Google Scholar 

  13. Roche Diagnostics. emPCR Method Manual: Lib-L LV GS FLX Titanium Series (Roche Applied Science, October 2009 (Rev. January 2010)).

  14. Roche Diagnostics. Sequencing Method Manual: GS FLX Titanium Series (Roche Applied Science, October 2009 (Rev. November 2010)).

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This study was supported by the Sixth Research Framework Programme of the European Union, a project on infections and cancers (INCA, LSHC-CT-2005-018704); the Karolinska Institutet Faculty funds for partial financing of new doctoral students (KID scholarship) to Z.Z.; and the Swedish Research Council Formas grant (FORMAS) to A.F.A.

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Authors and Affiliations



Z.Z. designed the Y adapters and qPCR assay, proposed Poisson statistics, conducted the experiments and drafted the manuscript; A.A., Ö.M., S.G. and H.N. assisted with the experiments and provided critical reviews; W.Y., L.E. and A.A. obtained funding and provided critical reviews; A.F.A. supervised the study.

Corresponding author

Correspondence to Zongli Zheng.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

454 Titanium library pooling calculation.xls: An example of calculation for 454 sample library pooling. (XLS 41 kb)

Supplementary Data 2

TrimYadapter.txt: A template file for user-customized Y adapter trimming. (XML 5 kb)

Supplementary Data 3

Yscheme.txt: User-defined MID sets for the 8 Y adapters. (TXT 0 kb)

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Zheng, Z., Advani, A., Melefors, Ö. et al. Titration-free 454 sequencing using Y adapters. Nat Protoc 6, 1367–1376 (2011).

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