Background Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity. Results A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5′ and 3′ ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system. Conclusions These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates. Electronic supplementary material The online version of this article (10.1186/s12896-019-0547-1) contains supplementary material, which is available to authorized users.

Multiplexed cloning of long DNA sequences is a valuable technique in many biotechnology applications, such as long-read genome sequencing and the creation of open reading frame (ORF) libraries. Long-adapter single-stranded oligonucleotide (LASSO) probes have shown promise as a tool to clone long DNA fragments. LASSO probes are molecular inversion probes (MIP) engineered with an adapter region of user-defined length, flanked between template-specific probe sequences. Herein, we demonstrate that the adapter length is a key feature of LASSO that influences the efficiency of gene capture and cloning. Furthermore, we applied a model based on Monte Carlo molecular simulation in order to study the relationship between the long-adapter length of LASSO and capture enrichment. Our results suggest that the adapter length is a factor that contributes to the free energy of target–probe interaction, thereby determining the efficiency of capture. The results indicate that LASSOs with extremely long adapters cannot capture the targets well. They also suggest that targets of different lengths may prefer adapters of different lengths.