Can Long-Range PCR Be Used to Amplify Genetically Divergent Mitochondrial Genomes for Comparative Phylogenetics? A Case Study within Spiders (Arthropoda: Araneae)

Abstract

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.

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale

“…This would increase taxonomic and phylogenetic resolution and alleviate some disadvantages of short COI amplicons. However, it is challenging to develop truly universal primers to target mitochondrial genomes across a very wide range of taxonomic groups [71]. A straightforward way to achieve highly resolved phylogenies may be the combination of long rDNA amplicon sequencing with multiplex PCRs of short mitochondrial amplicons to amplify multiple mitochondrial DNA fragments [72].…”

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale

“…This would increase taxonomic and phylogenetic resolution and thus alleviate some disadvantages of short COI amplicons. However, it is challenging to develop truly universal primers to target mitochondrial genomes across a wide range of taxonomic groups [64]. Moreover, mitochondrial genomes will not allow cases of mitonuclear discordance to be identified.…”

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription

“…The small size and circular nature of the majority of animal mtDNAs make them accessible for low-budget taxonomic studies, given the availability of simple and inexpensive protocols. It is now possible to amplify the complete mtDNA using long-range PCR ( Burger et al, 2007 ), which combined with novel high-throughput sequencing technologies, provide access to mitogenomic data for groups considered “difficult-to-sequence” at very low cost and effort ( Kayal et al, 2012 ; Briscoe et al, 2013 ; Foox et al, 2015 ). To date, the mtDNA of 188 non-bilaterian animals has been sequenced, out of which 124 are cnidarians, mostly anthozoans.…”