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
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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.

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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.


  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


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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.

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    Supplementary Information

    Supplementary methods, figures, tables and references.

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    Supplementary dataset

    Supplementary sequence data.