采用诱导环化酶制备Illumina Mate-paired DNA文库

新一代测序技术(NGS)的文库制备方法在基因组的拼装中起着重要作用。但是NGS技术制备的普通DNA文库片段只有500 bp左右,难以满足复杂基因组的从头(de novo)拼装要求。三代测序技术的读长可以达到20 kb,但是其高错误率及测序成本过高使得其又不易推广。因此二代测序的Mate-paired文库制备技术一直在基因组的de novo拼装中扮演着非常重要的角色。目前主流的NGS平台Illumina制备的Mate-paired文库的片段范围只有2~5 kb,为了得到更长的可用于Illumina平台测序的Mate-paired文库,本研究首次整合并优化了Illumina和Roche/454两种测序平台的Mate-paired文库制备技术,采用诱导环化酶来提高基因组长片段DNA的环化效率,成功建立了20 kb Mate-paired文库制备技术,并已将该技术应用于人类基因组20 kb Mate-paired文库制备。该技术为Illumina平台制备长片段Mate-paired库提供了方法指导。

Illumina Mate-paired DNA Sequencing-library Preparation Using Enzyme Induction

Next generation sequencing(NGS)plays an important role in assembly projects.But the fragments of commons DNA library produced by NGS te chnology is only about 500 bp,which is difficult to assemble the complex genome(de novo).The read length of three generations of sequencing technology can reach 20 kb,but its high error rate and high sequencing cost make it difficult to popularize.Therefore,the Mate-paired library preparation technology of the second generation has been playing a very important role in the genomic de novo assembly.Now the mainstream NGS platform Illumina mate-paired library fragment range is only 2~5 kb.In order to get enough long reads data through Illumina platform,we developed a mate-paired protocol with the combination of Illumina sequencing technology and Roche sequencing technology.It improved the efficiency of long insert fragments of cyclization through enzyme induction.We tested this method by preparing a mate-paired library with an insert size of 20 kb from Human Genomic DNA.This technique provides a method guide for the preparation of long fragments mate-paired library for Illumina platform.