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

Lorenzo Tosi, Viswanadham Sridhara, Yunlong Yang, Dongli Guan, Polina Shpilker, Nicola Segata, H. Benjamin Larman, Biju Parekkadan

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article number0092
JournalNature biomedical engineering
Volume1
Issue number7
DOIs
StatePublished - Jun 30 2017

Fingerprint

Oligonucleotide Probes
Oligonucleotides
Cloning
Open Reading Frames
Organism Cloning
DNA
Genes
Genome
Clone Cells
Functional analysis
DNA sequences
Metagenome
Escherichia coli
Microbiota
Regulator Genes
Complementary DNA
Throughput
Costs and Cost Analysis
Costs

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Biotechnology
  • Biomedical Engineering
  • Medicine (miscellaneous)
  • Computer Science Applications

Cite this

Tosi, Lorenzo ; Sridhara, Viswanadham ; Yang, Yunlong ; Guan, Dongli ; Shpilker, Polina ; Segata, Nicola ; Larman, H. Benjamin ; Parekkadan, Biju. / Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions. In: Nature biomedical engineering. 2017 ; Vol. 1, No. 7.
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Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions. / Tosi, Lorenzo; Sridhara, Viswanadham; Yang, Yunlong; Guan, Dongli; Shpilker, Polina; Segata, Nicola; Larman, H. Benjamin; Parekkadan, Biju.

In: Nature biomedical engineering, Vol. 1, No. 7, 0092, 30.06.2017.

Research output: Contribution to journalArticle

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AU - Larman, H. Benjamin

AU - Parekkadan, Biju

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