Consequently, DNA sequence data might be obtained from specimens of natural history collections with NGS, where PCR-based approaches fail. In fact, the first step in NGS library preparation is fragmentation of the DNA. Although enrichment of the mitochondrial fraction by long range PCR will increase the chance of obtaining a complete mitogenome (and facilitate the use of multiple specimens if sequence tags are exploited ), it is not essential to NGS (e.g. An alternative is the hybridization capture approach, but this requires a priori sequence knowledge in order to design probes. Due to the often degraded state of the DNA, this by and large excludes the use of specimens from natural history collections. Nearly all traditional (Sanger) and still some NGS approaches for sequencing mitogenomes rely on long range PCR amplification. With NGS technologies, primer-directed amplification is no longer necessary and genome sequencing, mitogenomes in particular because of the small size and high-copy-number, has become fast and easy. Moreover, the throughput of traditional (Sanger) sequencing is limited and sequencing of larger (> 1 kb) fragments is often delayed by the development of internal primers ('primer walking'). physical isolation of mitochondria, cloning of large mitochondrial fragments, long range, simplex or multiplex PCR) was quite laborious. Sequencing mitogenomes has been quite cumbersome because enrichment of the mitochondrial fraction (e.g. Of a third species, Euhadra herklotsi, most of the mitogenome has been covered, so comparisons of mitochondrial gene arrangements could be made, but a complete sequence for that mitogenome is still missing. For over two decades, the complete mitogenomes of only two stylommatophorans, Cepaea nemoralis and Albinaria caerulea, had been known. A comparison between Illumina GAIIx and the Roche 454 platform suggests that the former might be more suited for de novo sequencing of mitogenomes.Īlthough NGS techniques advanced rapidly over the last years and sequencing of entire mitochondrial genomes (mitogenomes) has consequently become more common, knowledge about stylommatophoran ('terrestrial snails') mitogenomes seems to have advanced at a slower pace. 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. 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). obtusus (subfamily Ariantinae) and might be a synapomorphy for the family Helicidae. nemoralis (subfamily Helicinae), but is also shown to occur in C. The 'aberrant' rearrangement of tRNA-Thr and COIII compared to that of other Sigmurethra (and the majority of gastropods), is not unique for C. obtusus is identical to that reported for Cepaea nemoralis. Except for a swap between the position of tRNA-Pro and tRNA-Ala, the gene arrangement 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. A brief comparison is made between our Illumina GAIIx approach and a similar study that made use of the Roche 454-FLX platform. 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. The mitogenome was obtained with Illumina GAIIx shot gun sequencing. 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.
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