Application of full mitochondrial genome sequencing using 454 GS FLX pyrosequencing

Beskrivelse

Partial sequences of the mitochondrial genome have been a valuable tool in forensic and population genetics for many years. However, it is of highest interest to achieve complete mitochondrial genome sequences in a cost and time efficient manner. This is now possible using 2^nd generation DNA sequencing platforms. The aim of this study was to test the performance of DNA sequencing using emulsion PCR followed by pyrosequencing on the GS FLX platform from 454 Life Sciences.

Materials and Methods

The method was tested on samples from 10 Somali individuals. For each sample, the complete mitochondrial genome was amplified in two parallel PCRs. PCR products from each sample were pooled and a library was established by fragmentation and tagging of the DNA followed by pooling of all the samples. Adapter sequences recognisable by the primers used in later steps were ligated to each end of the DNA. The pooled samples were amplified by emulsion PCR. Overlapping sequences with lengths up to 400 bp were generated using pyrosequencing and assembled using the software provided by the manufacturer.

Results and Discussion

The obtained sequences were compared to partial sequences generated with either terminator sequencing of the control region (position 16,024 to 576) or SNP typing of the coding region using single base extension (49 SNPs). The cost for reagents were approximately 60€ per sample for high quality sequencing (coverage ranging from 10-30 times). In conclusion, the GS FLX platform provides a fast, accurate and cheap method for obtaining full mitochondrial DNA sequences.