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


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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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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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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Authors and Affiliations



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).

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