HiMAP: Robust phylogenomics from highly multiplexed amplicon sequencing

Abstract

High-throughput sequencing has fundamentally changed how molecular phylogenetic data sets are assembled, and phylogenomic data sets commonly contain 50- to 100-fold more loci than those generated using traditional Sanger sequencing-based approaches. Here, we demonstrate a new approach for building phylogenomic data sets using single-tube, highly multiplexed amplicon sequencing, which we name HiMAP (highly multiplexed amplicon-based phylogenomics) and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and postsequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously and requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically important agricultural insect pests. The resulting filtered data set (>150,000-bp concatenated alignment, ~20% missing character sites across all individuals and amplicons) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting data set provides into relationships of this diverse insect group.

Highly variable COI haplotype diversity between three species of invasive pest fruit fly reflects remarkably incongruent demographic histories

“…Also, Kunprom et al (Kunprom et al 2015) reported a strongly divergent COI lineage within B. correcta, matching earlier results based on the COI-3P region (Jamnongluk et al 2003). They attributed this anomaly to Wolbachia bacterial infection, but upon comparison of the published COI-3P sequences with our COI reference dataset (data not shown) we conclude this lineage to match a cluster that includes B. nigrotibialis, B. nigrifacia and B. nigrofemoralis, which share some of the same hosts as B. correcta and can be morphologically similar (Allwood et al 1999;Drew and Romig 2013). For maximum likelihood tree inference of the relationships of the target species and their closest relatives (based on (San Jose et al 2018a), we used unique haplotypes only and added B. nigrotibialis (Perkins), Zeugodacus tau (Walker), Z. synnephes (Hendel) and Z. choristus (May) to the alignments.…”

“…The effects of historical climatic change were not as severe in Southeast Asia as in temperate regions, with more continuous possibilities for gene-flow throughout the region which may have resulted in less strongly pronounced biogeographic patterns (Penny 2001;Cannon and Manos 2003). However, a high resolution (1,097 SNPs) study of the melon fly Z. cucurbitae revealed geographic structuring with six to ten clusters that were only partly detected using microsatellite data (Dupuis et al 2018a), and are not detected in our current study using COI. Despite being a widespread species occurring from Africa across Asia into the Pacific, we only found a single dominant haplotype.…”

“…Partly overlapping with this distribution, B. correcta can be found throughout most of mainland South-East Asia (Fig. 1B) (Drew and Romig 2013). Samples were collected between 2005 and 2017 ( Fig.…”

“…Some specimens were additionally used for genomic studies (e.g. (Dupuis et al 2018a), such flies were fully ground up to obtain higher concentration DNA extracts. DNA was extracted using the Qiagen (Valencia, CA) DNeasy animal blood and tissue kit, following manufacturer's protocols.…”

Fluidigm2PURC: Automated processing and haplotype inference for double‐barcoded PCR amplicons

“…The newer Fluidigm Juno system can also handle up to 192 samples in a single run, and multiplexing of primer pairs can allow for even higher throughput, provided that the primers do not interact during amplification. Double‐barcoded amplicons can also be generated by other means using approaches such as traditional or highly multiplexed PCR (e.g., Bybee et al., ; Dupuis et al., ).…”