The development of second generation sequencing technology has resulted in the rapid production of large volumes of sequence data for relatively little cost, thereby substantially increasing the quantity of data available for phylogenetic studies. Despite these technological advances, assembling longer sequences, such as that of entire mitochondrial genomes, has not been straightforward. Existing studies have been limited to using only incomplete or nominally intra-specific datasets resulting in a bottleneck between mitogenome amplification and downstream high-throughput sequencing. Here we assess the effectiveness of a wide range of targeted long-range PCR strategies, encapsulating single and dual fragment primer design approaches to provide full mitogenomic coverage within the Araneae (Spiders). Despite extensive rounds of optimisation, full mitochondrial genome PCR amplifications were stochastic in most taxa, although 454 Roche sequencing confirmed the successful amplification of 10 mitochondrial genomes out of the 33 trialled species. The low success rates of amplification using long-Range PCR highlights the difficulties in consistently obtaining genomic amplifications using currently available DNA polymerases optimised for large genomic amplifications and suggests that there may be opportunities for the use of alternative amplification methods.

Supplementary table1Taxonomic information and primer sequences for specimensSupplementary table 2Taxonomic information and primer properties for specimensSliding window analysis of nucleotide diversitySliding window plots of nucleotide diversity (pi) across all 13 mitochondrial protein coding genes and both ribosomal RNA genes of six Araneae taxa.
Sliding_window_spider_genes.pdfRoche 454 sequence quality plotFrequency plot of phred quality scores from the total number of reads obtained from 454 sequencing454_Quality_plot.pdf454 assembled contigsFasta files of contigs assembled from 454 GS FLX sequencing data