Rapid DNA-Seq to Achieve High Coverage Libraries from 1ng – 1 μg in 2 Hours.
Year: 2014
Type: article
Abstract: As next-generation sequencing technology progresses, the need for highly efficient, non-biased library generation for a wide variety of inputs has grown. High throughput sequencing operations require library preparation protocols which optimize many parameters, such as enzyme efficiency, low input flexibility and time efficiency without sacrificing the quality of downstream bioinformatics analysis. We've recently developed a highly efficient adapter ligation procedure that significantly increases the number of adapters bound to DNA insert. This should theoretically improve complexity, diversity, alignment and reduce the number of PCR duplicates in a library. Results: To test this, the enhanced adapter ligation step was used in our library prep protocol to prepare bacterial and human libraries along with currently established DNA-seq protocols. Final libraries were then analyzed on both Illumina MiSeq and HiSeq platforms, followed by coverage and library complexity analysis of the sequence data. Depth of coverage as a function of input material was shown to outperform regular ligation, especially in the 1 ng to 10 ng DNA input range. Normalized coverage as a function of GC content revealed no intrinsic bias when compared to older ligation and amplification methods. The enhanced adapter ligation and several new modifications in this new protocol allow users for the first time to build complete genomic DNA libraries in as little as 2 hours with low nanogram input material.
Source: Europe PMC (PubMed Central)
Institution: Bioo Scientific (United States)
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2252349919