Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.
Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.
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DOI：
10.1093/nar/gkq807
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发表时间：
2010-11
影响因子：
14.9
通讯作者：
Vogler AP
中科院分区：
文献类型：
--
作者：
Timmermans MJ;Dodsworth S;Culverwell CL;Bocak L;Ahrens D;Littlewood DT;Pons J;Vogler AP
线粒体基因组序列是植物遗传学的重要标记，但由于获得全长序列需要付出巨大的努力和成本，分类群取样仍然是零星的。在这里，我们展示了一种简单，具有成本效益的方法，使用454/Roche平台从合并的样品中测序编码线粒体基因的蛋白质的完整互补序列。在不需要昂贵的索引标签（“条形码”）的情况下实现了多路复用。该方法进行了试验与一套远程聚合酶链反应（PCR）片段从30种鞘翅目（甲虫）测序在1/16扇区的测序板。从合并的序列中产生长重叠群，测序深度范围为每个重叠群100 ×至100×。通过三个“诱饵”序列匹配通过常规PCR和桑格测序获得的线粒体基因组的不同部分，建立了单个重叠群的物种身份。这证明来自测序池的重叠群的组装是正确的。我们的研究产生了21个几乎完整和7个部分蛋白质编码线粒体基因组的序列。结合已有的25个分类群的序列，得到了鞘翅目基本亲缘关系的改进估计。该程序可以在物种水平上常规地用于线粒体基因组测序，以提供目前仅使用cox 1基因的改进的物种“条形码”。
Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.
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DOI：
10.1098/rsbl.2003.0025
发表时间：
2003-08-07
影响因子：
4.7
作者：
Hebert, PDN;Ratnasingham, S;deWaard, JR
通讯作者：
deWaard, JR
使用454技术从单个Haemonchus contortus（Nematoda）的完整线粒体基因组进行长PCR测序。
影响因子：
4.4
作者：
Jex, Aaron R.;Hu, Min;Littlewood, D. Timothy J.;Waeschenbach, Andrea;Gasser, Robin B.
通讯作者：
Gasser, Robin B.
影响因子：
3.7
作者：
Andersson, Anders F.;Lindberg, Mathilda;Jakobsson, Hedvig;Backhed, Fredrik;Nyren, Pal;Engstrand, Lars
通讯作者：
Engstrand, Lars
影响因子：
--
作者：
Hong, Mee Yeon;Jeong, Heon Cheon;Kim, Iksoo
通讯作者：
Kim, Iksoo
影响因子：
4.1
作者：
Friedrich, M;Muqim, N
通讯作者：
Muqim, N