Moreira Daniel A, Furtado Carolina, Parente Thiago E
Laboratório de Toxicologia Ambiental, DCB, Escola Nacional de Saúde Pública - ENSP, Fundação Oswaldo Cruz - FIOCRUZ, Rio de Janeiro, Brasil.
Divisão de Genética, Instituto Nacional do Cancer - INCA, Rio de Janeiro, Brasil.
Gene. 2015 Nov 15;573(1):171-5. doi: 10.1016/j.gene.2015.08.059. Epub 2015 Sep 3.
Mitochondrial genes and genomes have long been applied in phylogenetics. Current protocols to sequence mitochondrial genomes rely almost exclusively on long range PCR or on the direct sequencing. While long range PCR includes unnecessary biases, the purification of mtDNA for direct sequencing is not straightforward. We used total RNA extracted from liver and Illumina HiSeq technology to sequence mitochondrial transcripts from three fish (Ancistrus spp.) and assemble their mitogenomes. Based on the mtDNA sequence of a close related species, we estimate to have sequenced 92%, 95% and 99% of the mitogenomes. Taken the sequences together, we sequenced all the 13 protein-coding genes, two ribosomal RNAs, 22 tRNAs and the D-loop known in vertebrate mitogenomes. The use of transcriptomic data allowed the observation of the punctuation pattern of mtRNA maturation, to analyze the transcriptional profile, and to detect heteroplasmic sites. The assembly of mtDNA from transcriptomic data is complementary to other approaches and overcomes some limitations of traditional strategies for sequencing mitogenomes. Moreover, this approach is faster than traditional methods and allows a clear identification of genes, in particular for tRNAs and rRNAs.
线粒体基因和基因组长期以来一直应用于系统发育学。目前用于线粒体基因组测序的方案几乎完全依赖于长片段PCR或直接测序。虽然长片段PCR存在不必要的偏差，但用于直接测序的线粒体DNA纯化并不简单。我们使用从肝脏中提取的总RNA和Illumina HiSeq技术对三种鱼类（Ancistrus spp.）的线粒体转录本进行测序，并组装它们的有丝分裂基因组。基于一个近缘物种的线粒体DNA序列，我们估计已对有丝分裂基因组的92%、95%和99%进行了测序。将这些序列放在一起，我们对脊椎动物有丝分裂基因组中已知的所有13个蛋白质编码基因、两个核糖体RNA、22个转运RNA和D环进行了测序。转录组数据的使用使得能够观察线粒体RNA成熟的标点模式、分析转录谱并检测异质性位点。从转录组数据组装线粒体DNA与其他方法互补，克服了传统有丝分裂基因组测序策略的一些局限性。此外，这种方法比传统方法更快，并且能够清晰地鉴定基因，特别是对于转运RNA和核糖体RNA。
