Bioinformatics Centre, North-Eastern Hill University , Shillong, Meghalaya , India.
PeerJ. 2013 Nov 12;1:e207. doi: 10.7717/peerj.207. eCollection 2013.
Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don't contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.
吸虫包括大量寄生线虫（圆线虫）和扁形动物（吸虫和绦虫扁虫），它们具有临床重要性。使用先进的分子工具对寄生扁虫进行遗传特征分析是诊断和控制感染的核心。尽管核基因组具有适合物种鉴定和分子特征分析的遗传标记（例如核糖体（r）DNA 中的标记），但线粒体（mt）基因组始终为信息丰富的系统发育和流行病学研究提供了丰富的新型标记来源。在过去的十年中，寄生扁虫 mtDNA 基因组学取得了一些重要进展，尤其是肺吸虫、肝吸虫和肠吸虫。片形吸虫，通常称为巨型肠吸虫，是感染人类的最大的复殖吸虫之一，主要存在于亚洲，特别是印度次大陆。下一代测序（NGS）技术现在为短时间内高通量测序、组装和注释提供了机会。本文描述了一种针对 F. buski 的 mt 基因组高通量测序和生物信息学管道，重点介绍了短读 NGS 平台（如 Ion Torrent 和 Illumina）的实用性，这些平台通过创新的 PCR 引物设计方法以及组装方法，成功地对 mt 基因组进行了测序和组装。我们利用我们尚未发表的 F. buski 的 NGS 全基因组序列数据，并将其与 Fasciola hepatica mtDNA 的现有数据进行比较，将其作为 Fasciola hepatica mtDNA 的参考基因组，设计用于扩增 F. buski mt 基因组序列的精确和特异性引物。进行了长距离 PCR 以创建富含 mtDNA 序列的 NGS 文库。使用两种不同的 NGS 平台对 F. buski mt 基因组进行完整测序、组装和注释。肠道吸虫的完整 mt 基因组序列长 14118bp，是迄今为止测序的最短的吸虫线粒体基因组。非编码控制区由 tRNA-Gly 基因分为两部分，不包含串联重复或二级结构，这是吸虫控制区的典型特征。基因内容和排列与 Fasciola hepatica 相同。F. buski mtDNA 基因组与 Fasciola hepatica 非常相似，基因排列与其他吸虫相似。本研究首次报道了肠道吸虫的 mtDNA，这将有助于借助 mtDNA NGS 数据研究 Fasciolidae 分类学和系统发育。更重要的是，它将成为比较线粒体基因组学和吸虫寄生虫系统发育研究的资源。
