为建立一种新的解析异尖线虫线粒体基因组序列的方法,并解码两种异尖线虫线粒体DNA全长,收集深圳市辖区内超市、农贸市场、批发市场销售的海水鱼样品,根据形态学方法分离异尖线虫.首先利用两步法提取线虫线粒体DNA;随后对DNA进行酶切,电泳回收所需长度的DNA片段,加上接头序列进行文库制备和上机测序;最后分析下机reads,拼接出两种异尖线虫线粒体DNA全长.结果显示:共提取31条海水鱼线虫线粒体DNA,质量浓度为2～20 ng/μL;对6份候选异尖线虫线粒体深度测序,获得约3 Mb total reads/样品的数据量;采用SPAdes软件对各样品reads序列进行组装,获得两种GeneBank中尚未收录的异尖线虫线粒体DNA全长.本研究利用DNA高通量深度测序技术和生物信息学DNA组装技术,报道了两种GeneBank中未收录的异尖线虫线粒体DNA全长,丰富了已有线虫科线粒体基因组多样性研究基础数据,为异尖线虫乃至其他物种线粒体DNA解密工作提供了新的研究方法.
Decoding Two Mitochondrial DNA Complete Sequences of Anisakis nematodes in Marine Fish by High-throughput Sequencing
In order to establish a new method to interpret mitochondrial genome sequences of Anisakis nematodes and to decode two kinds of mitochondrial DNA complete sequences,marine fish samples were collected from supermarkets,wet markets and wholesale markets across Shenzhen City to isolate Anisakis using a morphological method.Mitochondrial DNA was firstly extracted from Anisakis using a two-step method and then was enzymatically digested,DNA fragments with required length were recovered by electrophoresis,and were added with linker sequences for cluster preparation and machine sequencing.Clean reads were analyzed to splice two kinds of mitochondrial DNA complete sequences of Anisakis.The results showed that 31 nematode mitochondrial DNA was extracted from marine fish,with mass concentration of 2-20 ng/mL;6 candidate mitochondria genes were further sequenced to obtain a data volume of about 3 Mb clean reads per sample;the reads sequence of each sample was assembled using SPAdes,and two kinds of mitochondrial DNA complete sequences of Anisakis that had not been included in GeneBank were obtained.In conclusion,two kinds of mitochondrial DNA complete sequences of Anisakis that had not been included in GeneBank were obtained using high-throughput sequencing and bioinformatics DNA assembly technology,enriching current data for researches on mitochondrial genome diversity of Nematodes,and contributing to decoding mitochondrial DNA of Anisakis and other species as a new method.
万方数据
维普
