Netherlands Centre for Biodiversity Naturalis, PO Box 9517, Leiden RA 2300, The Netherlands.
BMC Genomics. 2012 Mar 26;13:114. doi: 10.1186/1471-2164-13-114.
This study describes how the complete mitogenome of a terrestrial snail, Cylindrus obtusus (Draparnaud, 1805) was sequenced without PCRs from a collection specimen that had been in 70% ethanol for 8 years. The mitogenome was obtained with Illumina GAIIx shot gun sequencing. Although the used specimen was collected relatively recently and kept in a DNA-friendly preservative (not formalin as frequently used with old museum specimens), we believe that the exclusion of PCRs as facilitated by NGS (Next Generation Sequencing) removes a great obstacle in DNA sequencing of collection specimens. A brief comparison is made between our Illumina GAIIx approach and a similar study that made use of the Roche 454-FLX platform.
The mtDNA sequence of C. obtusus is 14,610 bases in length (about 0.5 kb larger than other stylommatophoran mitogenomes reported hitherto) and contains the 37 genes (13 protein coding genes, two rRNAs and 22 tRNAs) typical for metazoans. Except for a swap between the position of tRNA-Pro and tRNA-Ala, the gene arrangement of C. obtusus is identical to that reported for Cepaea nemoralis. The 'aberrant' rearrangement of tRNA-Thr and COIII compared to that of other Sigmurethra (and the majority of gastropods), is not unique for C. nemoralis (subfamily Helicinae), but is also shown to occur in C. obtusus (subfamily Ariantinae) and might be a synapomorphy for the family Helicidae.
Natural history collections potentially harbor a wealth of information for the field of evolutionary genetics, but it can be difficult to amplify DNA from such specimens (due to DNA degradation for instance). Because NGS techniques do not rely on primer-directed amplification (PCR) and allow DNA to be fragmented (DNA gets sheared during library preparation), NGS could be a valuable tool for retrieving DNA sequence data from such specimens. A comparison between Illumina GAIIx and the Roche 454 platform suggests that the former might be more suited for de novo sequencing of mitogenomes.
本研究描述了如何从已在 70%乙醇中保存 8 年的馆藏标本中，通过二代测序（NGS）而无需进行 PCR 来获得陆生蜗牛 Cylindrus obtusus（Draparnaud，1805）的完整线粒体基因组序列。线粒体基因组通过 Illumina GAIIx shot gun 测序获得。尽管所用标本是相对近期采集的，并保存在 DNA 友好型防腐剂中（不像旧博物馆标本那样经常使用福尔马林），但我们认为 NGS 排除 PCR 作为 DNA 测序的一个主要障碍。我们比较了 Illumina GAIIx 方法和使用 Roche 454-FLX 平台的类似研究。
C. obtusus 的 mtDNA 序列长 14610 个碱基（比迄今报道的其他石磺目线粒体基因组长约 0.5 kb），包含 37 个基因（13 个蛋白质编码基因、两个 rRNA 和 22 个 tRNA），这是后生动物的典型特征。除了 tRNA-Pro 和 tRNA-Ala 位置的交换外，C. obtusus 的基因排列与 Cepaea nemoralis 报道的基因排列相同。与其他 Sigmurethra（和大多数腹足纲动物）相比，tRNA-Thr 和 COIII 的“异常”重排并非 C. nemoralis（ Helicinae 亚科）所特有，也发生在 C. obtusus（Ariantinae 亚科）中，可能是 Helicidae 科的一个 synapomorphy。
自然历史收藏为进化遗传学领域提供了丰富的信息，但从这些标本中扩增 DNA 可能很困难（例如由于 DNA 降解）。由于 NGS 技术不依赖于引物定向扩增（PCR），并且允许 DNA 片段化（在文库制备过程中 DNA 会被剪断），因此 NGS 可能是从这些标本中获取 DNA 序列数据的有用工具。Illumina GAIIx 和 Roche 454 平台的比较表明，前者可能更适合于线粒体基因组的从头测序。
