Computational Simulation of Adapter Length-Dependent LASSO Probe Capture Efficiency.

Jingqian Liu,Lorenzo Tosi,Alfred Tamayo,Syukri Shukor,Biju Parekkadan,Wilma K Olson,Vikas Nanda,Shuxiang Li,Benjamin Larman

doi:10.3390/biom9050199

Abstract

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.

Highlights

There is a dramatic asymmetry between genomic sequence information and the functional interpretation of that data, in part due to a lack of cost-efficient techniques for synthesizing stretches of DNA that are longer than 400 base pairs
The mature probe is comprised of a linear single oligonucleotide consisting of an adapter sequence between two probe arms that are complementary to the two ends of a target DNA fragment, a design that is similar to traditional molecular inversion probes (MIP) used for single nucleotide polymorphism analysis, except that the adapter length (AL)
This study evaluated the adapter region of a Long-adapter single-stranded oligonucleotide (LASSO) probe, both

Summary

Introduction

There is a dramatic asymmetry between genomic sequence information and the functional interpretation of that data, in part due to a lack of cost-efficient techniques for synthesizing stretches of DNA that are longer than 400 base pairs (bp). Probes were developed as a promising tool to fill this technological gap by cloning long genomic DNA targets in a parallel and multiplexed manner [1]. The mature probe is comprised of a linear single oligonucleotide consisting of an adapter sequence between two probe arms that are complementary to the two ends of a target DNA fragment, a design that is similar to traditional molecular inversion probes (MIP) used for single nucleotide polymorphism analysis, except that the adapter length (AL). LASSO probes have been shown to be far more effective than traditional MIPs at capturing DNA targets longer than 400 and up to 5000 bp [1,4,5]. The adapter regions of LASSO probes, which have been empirically optimized for given target sizes, warrant further investigation

Methods

Results

Conclusion