Abstract
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.
Highlights
Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost
We further demonstrate the benefits of using Tn5-059 in human genome sequencing including more uniform coverage and less sensitivity to DNA input amount variation
Random mutagenesis library of Tn5 transposase that aims to cover the entire coding region is constructed such that every mutant has approximately 1 to 6 random mutations
Summary
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 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. 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
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