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Genome of Datura stramonium: Ticuman grandparent
DNA extraction, genomic library preparation and sequencingTo obtain a high-quality de novo assembly, we combined data generated from short insert paired-end libraries from Illumina sequencing, with long read sequencing by PacBio Sequel II sequencing. First, gDNA was extracted from the two individuals. gDNA was isolated from fresh leaves with a modified CTAB mini-prep protocol (Doyle and Doyle 1987). The total amount of gDNA was measured using Qubit dsDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, USA). A total of 200 ng of gDNA were used for library preparation. Libraries were sheared on the Covaris and then prepped for 150PE (paired-end) Illumina HiSeq 4000 sequencing using the Kapa Hyper prep Illumina library prep kits. Final libraries were visualized on the Agilent Fragment Analyzer, then quantified and pooled equimolar with Kapa qPCR Illumina library quant Universal Kits. Demultiplexing was then performed with the Illumina bcl2fastq v2.19 software and returned in fastq format. The PacBio (Pacific Biosciences) was performed by taking 20 ug of gDNA into SMRTbell library preparation for long-insert libraries with the PacBio Express Template preparation kit, followed by size selection at 15kb on the Sage BluPippin. Libraries were then run on 10-hour movies on a PacBio Sequel using v2.0 chemistry. The sequencing and libraries preparation for both sequencing platforms was carried out in the QB3 Functional Genomics and Vincent J. Coates Sequencing Laboratories at the University of California, Berkeley.
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SUB7234150
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