An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899.
An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899.
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巨型肠fluke的完整线粒体基因组的下一代测序和注释的集成管道，fascioloppis buski（Lankester，1857年），1899年。
作者：
Biswal DK;Ghatani S;Shylla JA;Sahu R;Mullapudi N;Bhattacharya A;Tandon V
蠕虫包括寄生线虫（蛔虫）和扁形虫（吸虫和绦虫），数量丰富，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表征对于感染的诊断和控制至关重要。尽管核基因组包含适合物种鉴定和分子表征的遗传标记（例如，核糖体（r）DNA），但线粒体（mt）基因组始终为信息系统学和流行病学研究提供丰富的新型标记来源。在过去的十年中，蠕虫的线粒体DNA基因组学取得了一些重要进展，特别是肺吸虫、肝吸虫和肠吸虫。布斯基姜片吸虫（Fasciolopsisbuski），通常被称为巨型肠吸虫，是感染人类的最大的双殖纲吸虫之一，主要发现于亚洲，特别是印度次大陆。新一代测序 (NGS) 技术现在提供了在短时间内进行高通量测序、组装和注释的机会。在此，我们描述了用于 F.buski 的 mt 基因组学的高通量测序和生物信息学流程，强调了 Ion Torrent 和 Illumina 等短读长 NGS 平台在使用 PCR 引物设计和组装的创新方法成功测序和组装 mt 基因组方面的效用。我们利用 F.buski 的 NGS 全基因组序列数据（迄今为止尚未发表），并将其与可用的肝片形吸虫 mtDNA 数据进行比较，作为参考基因组，设计精确且特异的引物，用于扩增 F.buski 的 mt 基因组序列。进行长程 PCR 以创建富含 mt DNA 序列的 NGS 文库。采用两种不同的 NGS 平台对 F.buski mt 基因组进行完整测序、组装和注释。肠吸虫完整的mt基因组序列由14,118 bp组成，是迄今为止测序的最短的吸虫线粒体基因组。非编码控制区被 tRNA-Gly 基因分成两部分，并且不包含吸虫控制区典型的串联重复或二级结构。基因内容和排列与肝镰刀菌相同。 F.buski mtDNA 基因组与肝吸虫非常相似，并且具有与其他吸虫相似的基因顺序。我们的团队在此首次报道了肠吸虫的 mtDNA，这将有助于借助 mtDNA NGS 数据研究片形吸虫科的分类学和系统学。更重要的是，它将作为比较线粒体基因组学和吸虫寄生虫系统研究的资源。
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.
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