TY - JOUR
T1 - Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions
AU - Tosi, Lorenzo
AU - Sridhara, Viswanadham
AU - Yang, Yunlong
AU - Guan, Dongli
AU - Shpilker, Polina
AU - Segata, Nicola
AU - Larman, H. Benjamin
AU - Parekkadan, Biju
N1 - Publisher Copyright:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
PY - 2017/6/30
Y1 - 2017/6/30
N2 - 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.
AB - 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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U2 - 10.1038/s41551-017-0092
DO - 10.1038/s41551-017-0092
M3 - Article
C2 - 29152409
AN - SCOPUS:85029958503
SN - 2157-846X
VL - 1
JO - Nature biomedical engineering
JF - Nature biomedical engineering
IS - 7
M1 - 0092
ER -