Article

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

  • Nature Biomedical Engineering 1, Article number: 0092 (2017)
  • doi:10.1038/s41551-017-0092
  • Download Citation
Received:
Accepted:
Published online:

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.

  • Subscribe to Nature Biomedical Engineering for full access:

    $99

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    , , & Ten years of next-generation sequencing technology. Trends Genet. 30, 418–426 (2014).

  2. 2.

    , , , & Analyzing genes using closing and replicating circles. Trends Biotechnol. 24, 83–88 (2006).

  3. 3.

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

  4. 4.

    , , , & Massively parallel exon capture and library-free resequencing across 16 genomes. Nat. Methods 6, 315–316 (2009).

  5. 5.

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

  6. 6.

    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).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

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

Author notes

    • Lorenzo Tosi
    • , Viswanadham Sridhara
    •  & Yunlong Yang

    These authors contributed equally to this work.

Affiliations

  1. Department of Surgery, Center for Surgery, Innovation and Bioengineering, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, Massachusetts 02114, USA.

    • Lorenzo Tosi
    • , Viswanadham Sridhara
    • , Yunlong Yang
    • , Dongli Guan
    • , Polina Shpilker
    •  & Biju Parekkadan
  2. Centre for Integrative Biology, University of Trento, Trento 38123, Italy.

    • Nicola Segata
  3. Division of Immunology, Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21205, USA.

    • H. Benjamin Larman
  4. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA.

    • Biju Parekkadan
  5. Department of Biomedical Engineering, Rutgers University and the Department of Medicine, Rutgers Biomedical and Health Sciences, Piscataway, New Jersey 08854, USA.

    • Biju Parekkadan

Authors

  1. Search for Lorenzo Tosi in:

  2. Search for Viswanadham Sridhara in:

  3. Search for Yunlong Yang in:

  4. Search for Dongli Guan in:

  5. Search for Polina Shpilker in:

  6. Search for Nicola Segata in:

  7. Search for H. Benjamin Larman in:

  8. Search for Biju Parekkadan in:

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.

Competing interests

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

Corresponding authors

Correspondence to H. Benjamin Larman or Biju Parekkadan.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary methods, figures, tables and references.

Excel files

  1. 1.

    Supplementary dataset

    Supplementary sequence data.