Nebnext ultra iitmdna library prep kit

Total genomic DNA was extracted from ∼20 mg of leaf samples by a modified CTAB method (Doyle and Doyle, 1987 ). Approximately 5 μg of purified genomic DNA was used to construct shotgun libraries with a TruSeq DNA Sample Prep Kit following the manufacturer’s instructions (NEBNext^® Ultra IITMDNA Library Prep Kit for Illumina^®). Paired-end sequencing from both ends of 150 bp fragments was performed on an Illumina HiSeq X Ten platform (Illumina, San Diego, CA, United States) at BGI (Wuhan, China) to generate approximately 3 Gb data for each individual sample. Raw reads were filtered to remove adaptors and low quality reads using the NGS QC Toolkit (Patel and Jain, 2012 (link)) with default parameters.
The plastomes were de novo assembled from the genome skimming data using the GetOrganelle toolkit (Jin et al., 2020 (link)). The complete plastome of C. hainanensis (NC_042392) was used as the reference. Plastid genes were annotated using PGA (Qu et al., 2019 (link)) and coupled with manual adjustment in Geneious v8.0.2 (Kearse et al., 2012 (link)). The tRNAs were checked with tRNAscan-SE v2.0.3 (Lowe and Chan, 2016 (link)). Final plastid genome map was drawn using OGDRAW (Lohse et al., 2013 (link)).

Total genomic DNA was either extracted from silica-gel dried leaves using a modified CTAB method (Doyle and Doyle, 1987 ) or from herbarium specimens using the DNeasy Plant Mini Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Genomic DNA was then sheared into ca. 300 bp fragments, which were used for library construction following standard protocols (NEBNext^® Ultra IITMDNA Library Prep Kit for Illumina^®). Sequencing with 2 × 150 bp paired-end reads was conducted to generate approximately 2 Gb data for each accession using an Illumina HiSeq 2000 platform (Illumina, San Diego, CA, United States) at BGI Genomics (Shenzhen, Guangdong, China).
PCR mixtures and procedures for the amplification of ITS (including partial 18S, ITS1, 5.8S, ITS2, and partial 26S) and ETS followed those described in Chen et al. (2016a) (link), using primer pairs of 17SE and 26SE (Sun et al., 1994 (link)), and ETS-B (Beardsley and Olmstead, 2002 (link)) and 18S-IGS (Baldwin and Markos, 1998 (link)) for ITS and ETS, respectively. The PCR products were purified and sequenced by the Sangon Biotech (Shanghai, China) on an ABI 3730xl DNA Analyzer (Applied Biosystems, CA, United States).

The extracted DNA was sent to a sequencing company for automatic sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BIolabs) [80 (link)]. DNA extracts were quantified and sheared into approximately 500 base pair (bp) fragments for library construction using standard protocols (NEBNext Ultra IITMDNA Library Prep Kit for Illumina). Paired-end sequencing from both ends of 150 bp fragments was performed on the Illumina HiSeq X Ten platform at the Molecular Biology Experiment Center, Germplasm Bank of Wild Species in Southwest China, to generate no less than 2 GB data for each individual.
The paired-end reads were filtered using the GetOrganelle pipeline (https://github.com/Kinggerm/GetOrganelle) to obtain plastid-like reads [81 (link)] and then assembled using SPAdes version 3.10 [82 (link)]. A complete circular assembly graph was checked and further extracted using Bandage version 0.8.1 [83 (link)]. The genomes were automatically annotated using CpGAVAS [84 (link)], PGA (https://github.com/quxiaojian/PGA), and then manually adjusted using Geneious version 9.1.7 [85 (link)]. The chloroplast sequences generated in this study have been submitted to GenBank (Table S1). Circular genome maps of all 14 plastomes were also obtained using the Organellar Genome DRAW (OGDRAW) tool [86 (link)].