Improved genome sequencing using an engineered transposase
| Autor: | Igor Goryshin, Amirali Kia, Niall Anthony Gormley, Trina Faye Osothprarop, Michelle Stephenson, Erin Bomati, Christian Gloeckner, Molly He |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Sequence analysis 030106 microbiology Transposases Bacterial genome size Biology Protein Engineering Genome Sensitivity and Specificity DNA sequencing 03 medical and health sciences chemistry.chemical_compound Transposase Exome sequencing Genetics Chromosome Mapping High-Throughput Nucleotide Sequencing Reproducibility of Results DNA Sequence Analysis DNA AT Rich Sequence 030104 developmental biology chemistry Human genome Biotechnology Research Article |
| Zdroj: | BMC Biotechnology |
| ISSN: | 1472-6750 |
| Popis: | 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 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. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0326-1) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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