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

Nature Biomedical Engineering volume 1, Article number: 0092 (2017) Cite this article

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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.

DNA sequencing has created an unprecedented wealth of biological information1. With dramatic advances in parallelization, ‘reading’ DNA sequences has now become orders of magnitude more efficient and cost-effective than ‘writing’ them (for instance, synthesizing or cloning sequences of interest). Consequently, a bottleneck has formed between our knowledge of DNA sequences and our understanding of their functional significance. Massively parallel technologies enabling the synthesis and cloning of long DNA sequences are therefore required to bridge the widening gap from sequence to significance.

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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.

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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.).

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Author notes

  1. Lorenzo Tosi, Viswanadham Sridhara and Yunlong Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, Center for Surgery, Innovation and Bioengineering, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, 02114, Massachusetts, 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, 21205, Maryland, USA

    H. Benjamin Larman

  4. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA

    Biju Parekkadan

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

    Biju Parekkadan

Authors
  1. Lorenzo Tosi
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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.

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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)

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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

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