Lounsberry Z T, Brown S K, Collins P W, Henry R W, Newsome S D, Sacks B N
Mammalian Ecology and Conservation Unit, Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, One Shields Avenue/Old Davis Rd., Davis, CA, 95616, USA.
Santa Barbara Museum of Natural History, 2559 Puesta Del Sol, Santa Barbara, CA, 93105, USA.
Mol Ecol Resour. 2015 Jul;15(4):893-902. doi: 10.1111/1755-0998.12365. Epub 2015 Jan 13.
Use of complete mitochondrial genomes (mitogenomes) can greatly increase the resolution achievable in phylogeographic and historical demographic studies. Using next-generation sequencing methods, it is now feasible to efficiently sequence mitogenomes of large numbers of individuals once a reference mitogenome is available. However, assembling the initial mitogenomes of nonmodel organisms can present challenges, for example, in birds, where mtDNA is often subject to gene rearrangements and duplications. We developed a workflow based on Illumina paired-end, whole-genome shotgun sequencing, which we used to generate complete 19-kilobase mitogenomes for each of three species of North Pacific albatross, a group of birds known to carry a tandem duplication. Although this duplication had been described previously, our procedure did not depend on this prior knowledge, nor did it require a closely related reference mitogenome (e.g. a mammalian mitogenome was sufficient). We employed an iterative process including de novo assembly, reference-guided assembly and gap closing, which enabled us to detect duplications, determine gene order and identify sequence for primer positioning to resolve any mitogenome ambiguity (via minimal targeted Sanger sequencing). We present full mtDNA annotations, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes, a control region and a duplicated feature for all three species. Pairwise comparisons supported previous hypotheses regarding the phylogenetic relationships within this group and occurrence of a shared tandem duplication. The resulting mitogenome sequences will enable rapid, high-throughput NGS mitogenome sequencing of North Pacific albatrosses via direct reference-guided assembly. Moreover, our approach to assembling mitogenomes should be applicable to any taxon.
使用完整的线粒体基因组（线粒体基因组）可以大大提高系统地理学和历史种群统计学研究中的分辨率。利用下一代测序方法，一旦有了参考线粒体基因组，现在就可以有效地对大量个体的线粒体基因组进行测序。然而，组装非模式生物的初始线粒体基因组可能会带来挑战，例如在鸟类中，线粒体DNA经常发生基因重排和重复。我们开发了一种基于Illumina双端全基因组鸟枪法测序的工作流程，用于为北太平洋三种信天翁（一种已知携带串联重复的鸟类群体）中的每一种生成完整的19千碱基线粒体基因组。尽管这种重复之前已经被描述过，但我们的方法并不依赖于这些先验知识，也不需要密切相关的参考线粒体基因组（例如哺乳动物的线粒体基因组就足够了）。我们采用了一个迭代过程，包括从头组装、参考引导组装和缺口闭合，这使我们能够检测重复、确定基因顺序并识别引物定位的序列，以解决任何线粒体基因组的模糊性（通过最小靶向桑格测序）。我们给出了所有三个物种完整的线粒体DNA注释，包括22个tRNA、2个rRNA、13个蛋白质编码基因、一个控制区和一个重复特征。成对比较支持了之前关于该群体内系统发育关系和共享串联重复发生的假设。由此产生的线粒体基因组序列将通过直接参考引导组装实现北太平洋信天翁线粒体基因组的快速、高通量NGS测序。此外，我们组装线粒体基因组的方法应该适用于任何分类群。
