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.

中文翻译：

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LASSO探针组装和长时间多重克隆的定量评估。

背景技术长接头单链寡核苷酸（LASSO）探针被开发为大规模平行克隆千碱基长的基因组DNA序列的新型工具。LASSO可将当前DNA挂锁探针技术的捕获长度限制从大约150 bps显着提高到几kbps。高通量LASSO捕获涉及数千个探针的并行组装。但是，使用凝胶电泳技术无法将畸形的探针与正确形成的探针区分开。因此，我们使用了下一代测序（NGS）来评估LASSO探针组装的效率以及它与DNA捕获和扩增的性质之间的关系。此外，我们使用经典的分子生物学技术为探针特异性进行定性和定量评估，引入了简化的单靶LASSO方案。结果使用两种不同的探针组装反应条件组装了靶向3164种独特的大肠杆菌ORF的LASSO探针库，DNA浓度相差40倍。独特的探针序列位于5'和3'末端的前50 bps之内，因此我们使用配对末端NGS评估探针库的质量。正确映射的读对（代表正确形成的探针）分别占总读的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〜80％和〜20％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。仅使用组装有较低DNA浓度的探针进行捕获时，才能观察到LASSO靶标比非靶标显着富集。此外，半定量聚丙烯酰胺凝胶电泳显示，在简化的系统中，LASSO捕获的信噪比约为10倍。结论这些结果表明，与探针组装深度富集相比，LASSO探针对靶序列的覆盖率更能预测成功的捕获。同时，这些结果表明，探针装配反应中关键步骤的DNA浓度会显着影响探针的形成。此外，我们表明，与PAGE（聚丙烯酰胺凝胶电泳）耦合的简化LASSO捕获方案具有高度的特异性，并且更适合小规模的LASSO方法，例如筛选新型探针和模板。结论这些结果表明，与探针组装深度富集相比，LASSO探针对靶序列的覆盖率更能预测成功的捕获。同时，这些结果表明，探针装配反应中关键步骤的DNA浓度会显着影响探针的形成。此外，我们表明，与PAGE（聚丙烯酰胺凝胶电泳）耦合的简化LASSO捕获方案具有高度的特异性，并且更适合小规模的LASSO方法，例如筛选新型探针和模板。结论这些结果表明，与探针组装深度富集相比，LASSO探针对靶序列的覆盖率更能预测成功的捕获。同时，这些结果表明，探针装配反应中关键步骤的DNA浓度会显着影响探针的形成。此外，我们表明，与PAGE（聚丙烯酰胺凝胶电泳）耦合的简化LASSO捕获方案具有高度的特异性，并且更适合小规模的LASSO方法，例如筛选新型探针和模板。