tailieunhanh - Improved genome sequencing using an engineered transposase

Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. | Kia et al. BMC Biotechnology 2017 17 6 DOI S12896-016-0326-1 BMC Biotechnology RESEARCH ARTICLE Open Access I CrossMark Improved genome sequencing using an engineered transposase Amirali Kia1 Christian Gloeckner33 Trina Osothprarop1 Niall Gormley2 Erin Bomati1 Michelle Stephenson1 Igor Goryshin4 and Molly Min He1 Abstract Background Next-generation sequencing NGS has transformed genomic research by reducing turnaround time and cost. However no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods transposed-based Nextera has a slight insertion bias. Results Here we present the discovery of a mutant transposase Tn5-059 with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes which leads to less sequencing and lower cost per genome. In addition when used for human exomes Tn5-059 delivers consistent library insert size over a range of input DNA allowing up to a tenfold variance from the 50 ng input recommendation. Conclusions Enhanced DNA input tolerance ofTn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059 and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling. Background Library construction plays an important role for .