Improved genome sequencing using an engineered transposase

BMC Biotechnology

Research Article

Molly Min He¹  Erin Bomati¹  Amirali Kia¹  Michelle Stephenson¹  Trina Osothprarop¹  Igor Goryshin²  Christian Gloeckner³  Niall Gormley⁴ 

[1] Department of Protein Engineering, Illumina Inc, 5200 Illumina Way, San Diego, CA, USA;Illumina Inc, 5602 Research Park Blvd., Suite 200, Madison, WI, USA;NEO New Oncology GmbH, Gottfried-Hagen-Str. 20, 51105, Cologne, Germany;Technology Development, Illumina Inc, Little Chesterford, CB10 1XL, Nr Saffron Walden, Essex, UK;

关键词: Uniform Coverage;    Fragment Size Distribution;    Bias Plot;    Library Preparation Method;    Single Tube Reaction;   

DOI  :  10.1186/s12896-016-0326-1

 received in 2016-06-24, accepted in 2016-12-23,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundNext-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.ResultsHere 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.ConclusionsEnhanced DNA input tolerance of Tn5-059 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.

【 授权许可】

CC BY   
© The Author(s). 2017