Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions

Abstract

As the catalogue of sequenced genomes and metagenomes continues to grow, massively parallel approaches for the comprehensive and functional analysis of gene products and regulatory elements are becoming increasingly valuable. Current strategies to synthesize or clone complex libraries of DNA sequences are limited by the length of the DNA targets, throughput and cost. Here, we show that long-adapter single-strand oligonucleotide (LASSO) probes can capture and clone thousands of kilobase DNA fragments in a single reaction. As proof of principle, we simultaneously cloned over 3,000 bacterial open reading frames (ORFs) from Escherichia coli genomic DNA (spanning 400- to 5,000-bp targets). Targets were enriched up to a median of around 60-fold compared with non-targeted genomic regions. At a cutoff of three times the median non-target reads per kilobase of genetic element per million reads, around 75% of the targeted ORFs were successfully captured. We also show that LASSO probes can clone human ORFs from complementary DNA, and an ORF library from a human-microbiome sample. LASSO probes could be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Synthesis of the DNA LASSO probe components.
Figure 2: Single ORF target capture with LASSO probes.
Figure 3: Functional assessment of a LASSO-captured ORF target.
Figure 4: Comparison of ORFeome capture using LASSO or MIP probe libraries.
Figure 5: Multiplex capture and sequencing of an E. coli ORFeome library.
Figure 6: LASSO-based cloning and characterization of full-length ORFs from human cDNA or DNA isolated from a human microbiome sample.

References

  1. 1

    Van Dijk, E. L., Auger, H., Jaszczyszyn, Y. & Thermes, C. Ten years of next-generation sequencing technology. Trends Genet. 30, 418–426 (2014).

    CAS  Article  Google Scholar 

  2. 2

    Nilsson, M., Dahl, F., Larsson, C., Gullberg, M. & Stenberg, J. Analyzing genes using closing and replicating circles. Trends Biotechnol. 24, 83–88 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Dahl, F. et al. Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc. Natl Acad. Sci. USA 104, 9387–9392 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Turner, E. H., Lee, C., Ng, S. B., Nickerson, D. A. & Shendure, J. Massively parallel exon capture and library-free resequencing across 16 genomes. Nat. Methods 6, 315–316 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085–2088 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Landegren, U. et al. Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes. J. Mol. Recognit. 17, 194–197 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Hagerman, P. J. Flexibility of DNA. Annu. Rev. Biophys. Biophys. Chem. 17, 265–286 (1988).

    CAS  Article  Google Scholar 

  8. 8

    Krishnakumar, S. et al. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc. Natl Acad. Sci. USA 105, 9296–9301 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Shen, P. et al. Multiplex target capture with double-stranded DNA probes. Genome Med. 5, 50 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Shen, P. et al. High-quality DNA sequence capture of 524 disease candidate genes. Proc. Natl Acad. Sci. USA 108, 6549–6554 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    CAS  Article  Google Scholar 

  12. 12

    Tewhey, R. et al. Direct identification of hundreds of expression-modulating variants using a multiplexed reporter assay. Cell 165, 1519–1529 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Boeke, J. D. et al. The genome project—write. Science 353, 126–127 (2016).

    CAS  Article  Google Scholar 

  14. 14

    Bolger, A. M. et al. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS  Article  Google Scholar 

  15. 15

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  16. 16

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  17. 17

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Shriners Hospitals for Children (B.P. and L.T.), a Prostate Cancer Foundation Young Investigator award (H.B.L.), and National Institutes of Health Grants R01EB012521 (B.P.), K01DK087770 (B.P.) and 1U24AI118633 (H.B.L.).

Author information

Affiliations

Authors

Contributions

L.T., H.B.L. and B.P. conceived and designed the study. L.T., V.S., Y.Y., D.G., P.S. and N.S. performed the experiments, and analysed and interpreted the data. L.T., H.B.L. and B.P. wrote the manuscript.

Corresponding authors

Correspondence to H. Benjamin Larman or Biju Parekkadan.

Ethics declarations

Competing interests

A patent application on the technology has been filed (PCT/US2016/035919). The authors declare no other competing financial interests.

Supplementary information

Supplementary Information

Supplementary methods, figures, tables and references. (PDF 3963 kb)

Supplementary dataset

Supplementary sequence data. (XLSX 2241 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tosi, L., Sridhara, V., Yang, Y. et al. Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions. Nat Biomed Eng 1, 0092 (2017). https://doi.org/10.1038/s41551-017-0092

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing