Abstract
BackgroundNext-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface.ResultsThe surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates.ConclusionsWe described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.
Highlights
Next-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation
We believe that the NGS pipeline would be significantly improved if the library could be directly prepared on a flowcell surface, eliminating the need for upfront library construction
Overall process of tagmentation on polyacrylamide gel In order to perform the tagmentation on a solid surface rather than in solution, we first attached the Tn5 transposases on a surface
Summary
Next-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. After several cycles of PCR, the DNA library is ready to go through several quality control steps and load into the NGS instrument [3]. These steps typically take 8 to 10 h of hands-on work and expensive equipment is needed (e.g. Covaris). The Nextera kit improves this process by combining genome fragmentation and adaptor ligation into a single step, which is called tagmentation. Transposases used in the Nextera kit contain adaptors and when mixed with genomic DNA, they will shear the DNA and attach the adaptors to both ends of DNA fragments This process is very efficient and only takes a few minutes. Though the library preparation has been simplified by using tagmentation, PCR is still
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