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.

随着测序基因组和元基因组目录的不断增长，用于基因产物和调控元件的全面和功能分析的大规模并行方法变得越来越有价值。合成或克隆复杂的DNA序列文库的当前策略受到DNA靶标的长度，通量和成本的限制。在这里，我们显示长适配器单链寡核苷酸（LASSO）探针可以在单个反应中捕获和克隆数千个千碱基的DNA片段。作为原理证明，我们同时从大肠杆菌中克隆了3,000多个细菌开放阅读框（ORF）基因组DNA（跨越400-5,000 bp的靶标）。与非靶向基因组区域相比，靶标富集至中位值约60倍。在每百万个碱基每千个碱基的非目标碱基中位数的三倍的截断值下，成功捕获了约75％的目标ORF。我们还显示LASSO探针可以从互补DNA克隆人ORF，并从人微生物组样品中克隆ORF文库。LASSO探针可用于制备长读测序文库和大规模多重克隆。