Truseq pcr free library preparation

We used single specimen samples from four eusocial species (S. chacei, S. filidigitus, S. microneptunus, and S. regalis) and four noneusocial species (S. carpenteri, S. hoetjesi, S. kensleyi, and S. pandionis) for DNA extraction and low-coverage whole-genome sequencing. These species included three independent origins of eusociality, as well as two species from each of the four Synalpheus major clades (supplementary table S1, Supplementary Material online). The field collection protocol has been reported in Macdonald et al. (2006) . We extracted genomic DNA using several walking legs from alcohol-preserved specimens with Qiagen DNeasy Tissue Kits (Qiagen). Extracted DNA was quantified using a Qubit 3.0 Fluorometer with the dsDNA HS assay (ThermoFisher Scientific) and visualized on 2% agarose gels. We provided 1,500 ng of genomic DNA to Novogene (Chula Vista, CA) for TruSeq PCR-free library preparation (Illumina) and 150-bp pair-end sequencing on an Illumina NovaSeq to obtain at least 1× coverage according to published genome size (4.8–11.8 GB) (Jeffery et al. 2016 (link)). Low-coverage whole-genome sequencing reads from whole-cell extraction contain a high copy number of extranuclear sequences, and it has been shown to be an efficient and economical approach to assemble complete mitochondrial genomes (Dierckxsens et al. 2016 ).

High quality genomic data were extracted from ear notch biopsies for 130 sheep using a Nucleospin Tissue Kit and quality checked using an Agilent Tapestation 2200. For library preparation, 1 µg of gDNA was sheared to fragments of 450 bp mean size using a Covaris LE220 focused-ultrasonicator. DNA fragments were blunt ended, A-tailed, size selected and adapters ligated onto fragment ends according to Illumina TruSeq PCR-free library preparation kit protocol. Insert size on the libraries was evaluated using a PerkinElmer LapChip GX Touch with an HT DNA 1k/12K/HI SENS LabChip and HT DNA HI SENS Reagent Kit. Final library concentration was calculated by qPCR using a Roche LightCycler 480 and a Kapa Illumina Library Quantification kit and Standards. Then libraries were normalized to a loading concentration of 150 nM. All the library processing steps were carried out on Hamilton MicroLab STAR liquid handling robots coupled to BaseSpace Clarity LIMS X Edition. Libraries for all samples were loaded into a HiSeq X Flow cell v2.5, and clustered using an Illumina cBot2 Cluster Generation System. All libraries were sequenced on the HiSeqX to a mean coverage of 54X with 150 bp paired-end reads.