Abstract
BackgroundLong 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.ResultsA 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.ConclusionsThese 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.
Highlights
Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences
In an effort to improve upon padlock probe technology, we have previously shown that Long Adapter Single-Stranded Oligonucleotide (LASSO) probes improve the target capture size limitation of molecular inversion probes (MIPs), and demonstrated the massively multiplexed capture-by-circularization of kilobase long genomic regions from E. coli genomic DNA [5]
Since the sequence identities of LASSO probes cannot be discerned by standard molecular biology techniques such as electrophoretic mobility, we did not have a clear understanding of LASSO probe assembly efficiency and how that relates to successful captures downstream
Summary
Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. In an effort to improve upon padlock probe technology, we have previously shown that Long Adapter Single-Stranded Oligonucleotide (LASSO) probes improve the target capture size limitation of MIPs, and demonstrated the massively multiplexed capture-by-circularization of kilobase long genomic regions (up to ~ 4 kb) from E. coli genomic DNA [5]. Since the sequence identities of LASSO probes cannot be discerned by standard molecular biology techniques such as electrophoretic mobility, we did not have a clear understanding of LASSO probe assembly efficiency and how that relates to successful captures downstream. To this end, we set out to quantify LASSO probe assembly using next-generation sequencing techniques (NGS), and correlate those results to the success rate of LASSO capture. Our results confirm the critical nature of LASSO probe assembly and suggest how this step might be improved
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