Design: We quantified DNA for each sample using a Qubit fluorometer (High sensitivity kit, Life Technologies, Inc.) and sheared 2.8497 ng (139 ng mean) DNA to a target size of approximately 500600 bp by sonication (Q800 or Diagenode BioRuptor; Qsonica Inc.). The sheared DNA was used as input for a modified genomic DNA library preparation protocol (Kapa Hyper Prep Library Kit, Kapa Biosystems) that incorporated with-bead cleanup steps (Fisher et al. 2011) and a generic SPRI substitute (Rohland & Reich 2012, speedbeads hereafter), as described by Faircloth et al. (2014). We used TruSeq-style adapters during adapter ligation (Faircloth and Glenn, 2012), and PCR amplified 50% of the resulting library volume (15 ľL) using a reaction mix of 25 ľL HiFi HotStart polymerase (Kapa Biosystems), 2.5 ľL each of Illumina TruSeq i5 and i7 primers (5 ľM each) and 5 ľL double-distilled water (ddH20). We used the following thermal protocol: 98şC for 45 s; 13 cycles of 98şC for 15 s, 65şC for 30 s, 72şC for 60 s, and final extension at 72şC for 5 m. After rehydrating (in 23ľL pH 8 Elution Buffer (EB hereafter)) and purifying reactions using 1.0X speedbeads, we combined groups of eight libraries at equimolar ratios into enrichment pools having final concentrations of 74156 ng/ľL.We enriched each pool using a set of 2749 custom-designed probes (MYcroarray, Inc.) targeting 1510 UCE loci in Hymenoptera (see Faircloth et al. 2014). We followed library enrichment procedures for the MYcroarray MYBaits kit (Blumenstiel et al. 2010), except we used a 0.1X concentration of the standard MYBaits concentration, and added 0.7 ľL of 500 ľM custom blocking oligos designed against our custom sequence tags. We ran the hybridization reaction for 24 h at 65° C, subsequently bound all pools to streptavidin beads (MyOne C1; Life Technologies), and washed bound libraries according to a standard target enrichment protocol (Blumenstiel et al. 2010). We used the with-bead approach for PCR recovery of enriched libraries as described in Faircloth et al. (2014). We combined 15 ľL of streptavidin bead-bound, enriched library with 25 ľL HiFi HotStart Taq (Kapa Biosystems), 5 ľL of Illumina TruSeq primer mix (5 ľM each) and 5 ľL of ddH2O. We ran post-enrichment PCR using the following thermal profile: 98° C for 45 s; 18 cycles of 98° C for 15 s, 60° C for 30 s, 72° C for 60 s; and a final extension of 72° C for 5 m. We purified resulting reactions using 1.0X speedbeads, and we rehydrated the enriched pools in 22 ľL EB. We quantified 2 ľL of each enriched pool using a Qubit fluorometer (broad range kit). Enrichment was verified by amplifying seven UCE loci (for primers see Faircloth et al. 2014) targeted by the probe set. We set up a relative qPCR by amplifying two replicates of 1 ng of enriched DNA from each library at all seven loci and comparing those results to two replicates of 1 ng unenriched DNA for each library at all seven loci. We performed qPCR using a SYBRŽ FAST qPCR kit (Kapa Biosystems) on a Roche LightCycler 480. Following data collection, we computed the average of the replicate crossing point (Cp) values for each library at each amplicon, and we computed fold-enrichment values, assuming an efficiency of 1.78 and using the formula 1.78abs(enriched Cp ? unenriched Cp). We then created serial dilutions of each pool (1:200,000, 1:800,000, 1:1.000,000, 1:10.000,000) and performed qPCR library quantification, assuming an average library fragment length of 600 bp. Based on the size-adjusted concentrations estimated by qPCR, we pooled libraries at equimolar concentrations and size-selected for 250800 with a BluePippin (SageScience). The pooled libraries were sequenced using two partial lanes (both lanes included samples from other projects) of a 150-bp paired-end Illumina HiSeq 2500 run (U Cornell Genomics Facility).
Submitted by: National Museum of Natural History, Smithsonian
Study:
Phylogenomics of the ant subfamily Formicinae, based on UCE sequencesshow Abstracthide AbstractUltraconserved elements (UCEs) have been successfully used in phylogenomics for a variety of taxa, but their power in phylogenetic inference has not yet been extensively compared with traditional Sanger sequencing data sets. Moreover, UCE data on invertebrates, such as insects, is sparse. We compared the phylogenetic potency and informativeness of 959 UCE loci with a multi-locus data set of ten nuclear markers, by testing these two types of data and methods in their ability to resolve and date the evolutionary history of the ant subfamily Formicinae. Phylogenetic estimations show that UCEs are superior in resolving ancient and shallow relationships of formicine ants, demonstrated by increased node support and a more resolved phylogeny. Quantifications of phylogenetic informativeness further evidence a two-fold increase relative to the 10-gene data set. We were able to significantly improve formicine tribal classification based on our comprehensive UCE phylogeny for the group. Our divergence age estimations, using both UCE and Sanger data, indicate that crown-group formicine ants (104–117 Ma) are relatively older than previous studies have suggested. Biogeographic analyses infer the diversification of the subfamily as fairly spread out across all continents with no particular and apparent hub of cladogenesis. Much of the early history of the clade, however, remains uncertain due to ancient rapid divergence events unsolvable even with our genomic data. Our comparative study highlights the promise of UCEs for insect phylogenomics, as well as limitations, and will prove highly useful for the growing number of evolutionary biologists considering the transition from Sanger to next-generation sequencing approaches.
Library:
Name: Rossomyrmex anatolicus_T825
Instrument: Illumina HiSeq 2500
Strategy: WGS
Source: GENOMIC
Selection: Hybrid Selection
Layout: PAIRED
Runs:
1 run, 1.3M spots, 356.2M bases, 145.1Mb