Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

Abstract

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing.

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References 30

1. 1.
   Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition · 2010

   No notice
2. 2.
   HTSeq—a Python framework to work with high-throughput sequencing data · 2014

   No notice
3. 3.

   Ashley E. A., 2016 Towards precision medicine. Nat. Rev. Genet. 17: 507–522.

   Not in our library10.1038/nrg.2016.86

4. 4.

   Buenrostro J. D. Wu B. Litzenburger U. M. Ruff D. Gonzales M. L., 2015 Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523: 486–490.

   Not in our library10.1038/nature14590

5. 5.

   Davies D. R. Goryshin I. Y. Reznikoff W. S. Rayment I., 2000 Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science 289: 77–85.

   Not in our library10.1126/science.289.5476.77

6. 6.

   Esplin E. D. Oei L. Snyder M. P., 2014 Personalized sequencing and the future of medicine: discovery, diagnosis and defeat of disease. Pharmacogenomics 15: 1771–1790.

   Not in our library10.2217/pgs.14.117

7. 7.

   Giladi A. Amit I., 2017 Immunology, one cell at a time. Nature 547: 27–29.

   Not in our library10.1038/547027a

8. 8.

   Goryshin I. Y. Reznikoff W. S., 1998 Tn5 in vitro transposition. J. Biol. Chem. 273: 7367–7374.

   Not in our library10.1074/jbc.273.13.7367

9. 9.

   Goryshin I. Y. Miller J. A. Kil Y. V. Lanzov V. A. Reznikoff W. S., 1998 Tn5/IS50 target recognition. Proc. Natl. Acad. Sci. USA 95: 10716–10721.

   Not in our library10.1073/pnas.95.18.10716

10. 10.

    Grun D. Lyubimova A. Kester L. Wiebrands K. Basak O., 2015 Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525: 251–255.

    Not in our library10.1038/nature14966

11. 11.

    Hicks S. C. Teng M. Irizarry R. A., 2015 On the widespread and critical impact of systematic bias and batch effects in single-cell RNA-Seq data. bioRxiv Available at: https://pdfs.semanticscholar.org/aa12/953bdc4cbc2c01f9b1618c6d2818f5cb37c6.pdf.

    Not in our library

12. 12.

    Jaitin D. A. Kenigsberg E. Keren-Shaul H. Elefant N. Paul F., 2014 Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types. Science 343: 776–779.

    Not in our library10.1126/science.1247651

13. 13.

    Johnson R. C. Reznikoff W. S., 1983 DNA sequences at the ends of transposon Tn5 required for transposition. Nature 304: 280–282.

    Not in our library10.1038/304280a0

14. 14.

    Macosko E. Z. Basu A. Satija R. Nemesh J. Shekhar K., 2015 Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161: 1202–1214.

    Not in our library10.1016/j.cell.2015.05.002

15. 15.
    pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes · 2011

    No notice
16. 16.

    Nagano T. Lubling Y. Stevens T. J. Schoenfelder S. Yaffe E., 2013 Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature 502: 59–64.

    Not in our library10.1038/nature12593

17. 17.

    Paul F. Arkin Y. Giladi A. Jaitin D. A. Kenigsberg E., 2015 Transcriptional heterogeneity and lineage commitment in myeloid progenitors. Cell 163: 1663–1677.

    Not in our library10.1016/j.cell.2015.11.013

18. 18.

    Picelli S. Bjorklund A. K. Faridani O. R. Sagasser S. Winberg G., 2013 Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10: 1096–1098.

    Not in our library10.1038/nmeth.2639

19. 19.
    Tn5 transposase and tagmentation procedures for massively scaled sequencing projects · 2014

    No notice
20. 20.

    Picelli S. Faridani O. R. Björklund A. K. Winberg G. Sagasser S., 2014 b Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9: 171–181.

    Not in our library10.1038/nprot.2014.006

21. 21.

    Ramskold D. Luo S. Wang Y. C. Li R. Deng Q., 2012 Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30: 777–782.

    Not in our library10.1038/nbt.2282

22. 22.

    Reznikoff W. S., 2008 Transposon Tn5. Annu. Rev. Genet. 42: 269–286.

    Not in our library10.1146/annurev.genet.42.110807.091656

23. 23.

    Sasakawa C. Carle G. F. Berg D. E., 1983 Sequences essential for transposition at the termini of IS50. Proc. Natl. Acad. Sci. USA 80: 7293–7297.

    Not in our library10.1073/pnas.80.23.7293

24. 24.

    Shevchenko Y. Bouffard G. G. Butterfield Y. S. N. Blakesley R. W. Hartley J. L., 2002 Systematic sequencing of cDNA clones using the transposon Tn5. Nucleic Acids Res. 30: 2469–2477.

    Not in our library10.1093/nar/30.11.2469

25. 25.

    Velten L. Anders S. Pekowska A. Jarvelin A. I. Huber W., 2015 Single-cell polyadenylation site mapping reveals 3′ isoform choice variability. Mol. Syst. Biol. 11: 812.

    Not in our library10.15252/msb.20156198

26. 26.

    Velten L. Haas S. F. Raffel S. Blaszkiewicz S. Islam S., 2017 Human haematopoietic stem cell lineage commitment is a continuous process. Nat. Cell Biol. 19: 271–281.

    Not in our library10.1038/ncb3493

27. 27.

    Weinreich M. D. Gasch A. Reznikoff W. S., 1994 Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization. Genes Dev. 8: 2363–2374.

    Not in our library10.1101/gad.8.19.2363

28. 28.

    Wu T. D. Nacu S., 2010 Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26: 873–881.

    Not in our library10.1093/bioinformatics/btq057

29. 29.

    Zhou M. Reznikoff W. S., 1997 Tn5 transposase mutants that alter DNA binding specificity. J. Mol. Biol. 271: 362–373.

    Not in our library10.1006/jmbi.1997.1188

30. 30.

    Zhou M. Bhasin A. Reznikoff W. S., 1998 Molecular genetic analysis of transposase-end DNA sequence recognition: cooperativity of three adjacent base-pairs in specific interaction with a mutant Tn5 transposase. J. Mol. Biol. 276: 913–925.

    Not in our library10.1006/jmbi.1997.1579

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