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Titration-free 454 sequencing using Y adapters

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Abstract

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.

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Acknowledgements

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

  1. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden

    Zongli Zheng & Weimin Ye

  2. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

    Zongli Zheng, Öjar Melefors, Henrik Nordström & Lars Engstrand

  3. Swedish Institute for Infectious Disease Control, Solna, Sweden

    Abdolreza Advani, Öjar Melefors, Steve Glavas, Henrik Nordström & Lars Engstrand

  4. Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden.

    Anders F Andersson

Authors
  1. Zongli Zheng
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  2. Abdolreza Advani
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  3. Öjar Melefors
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  4. Steve Glavas
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  5. Henrik Nordström
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  6. Weimin Ye
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  7. Lars Engstrand
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  8. Anders F Andersson
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Contributions

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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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). https://doi.org/10.1038/nprot.2011.369

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