LASSO 探针组装和长读长片段多重克隆的定量评估。

Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning.

机构信息

Center for Surgery, Innovation, and Bioengineering, Department of Surgery, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, 51 Blossom Street, Boston, MA, 02114, USA.

Division of Immunology, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

出版信息

BMC Biotechnol. 2019 Jul 24;19(1):50. doi: 10.1186/s12896-019-0547-1.

DOI:10.1186/s12896-019-0547-1

PMID:31340783

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6657055/

Abstract

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.

摘要

背景

长接头单链寡核苷酸（LASSO）探针作为一种新的工具被开发出来，用于大规模平行克隆长达几千碱基对的基因组 DNA 序列。LASSO 将当前 DNA 套索探针技术的捕获长度限制从大约 150bp 显著提高到几千 bp。高通量 LASSO 捕获涉及数千个探针的并行组装。然而，使用凝胶电泳技术，无法区分畸形探针和正常形成的探针。因此，我们使用下一代测序（NGS）来评估 LASSO 探针组装的效率，以及它与 DNA 捕获和扩增的性质的关系。此外，我们引入了一种简化的单靶 LASSO 方案，使用经典分子生物学技术进行探针特异性的定性和定量评估。

结果

使用两种不同的探针组装反应条件，以 DNA 浓度相差 40 倍的方式，组装了一个靶向 3164 个独特大肠杆菌 ORF 的 LASSO 探针库。独特的探针序列位于 5'和 3'末端的前 50bp 内，因此我们使用配对末端 NGS 来评估探针库的质量。正确映射的读对，代表正确形成的探针，占总读取量的 10.81%和 0.65%，分别对应于较低和较高 DNA 浓度条件下总探针库的约 80%和 20%的覆盖率。随后，我们使用单端 NGS 来关联探针组装效率和捕获质量。只有在使用较低 DNA 浓度组装的探针进行捕获时，才能观察到 LASSO 靶标相对于非靶标的显著富集。此外，简化系统中的聚丙烯酰胺凝胶电泳（PAGE）显示出 LASSO 捕获的信号与噪声比约为 10 倍。

结论

这些结果表明，LASSO 探针对目标序列的覆盖率比探针组装深度富集更能预测成功捕获。同时，这些结果表明，探针组装反应中一个关键步骤的 DNA 浓度显著影响探针的形成。此外，我们还表明，与 PAGE （聚丙烯酰胺凝胶电泳）耦合的简化 LASSO 捕获方案具有高度特异性，并且更适合于小规模 LASSO 方法，例如筛选新型探针和模板。