title: RADseq of  Brontispa longissima: HN13
design: RAD-seq libraries were constructed following a modified protocol. Genomic DNA (0.11 g from accession samples) was extracted, and the restriction endonuclease EcoRI (New England Biolabs) was used to digest the genome followed by heat inactivation of the enzyme. The EcoRI cut site of each sample was ligated with a barcoded P1 adapters (complementing the EcoRI-cut DNA gap). These adapters contain forward amplification and Illumina sequencing primer sites, as well as a nucleotide barcode 4 or 8 bp long for sample identification. The adapter-ligated fragments were subsequently pooled, randomly sheared, and size-selected. DNA was then ligated to a second adapter (P2), a Y adapter that has divergent ends. The reverse amplification primer is unable to bind to P2 unless the complementary sequence was filled in during the first round of forward elongation originating from the P1 amplification primer. The structure of this adapter ensures that only P1 adapter-ligated RAD tags was amplified during the final PCR amplification step. During the QC step, Agilent 2100 Bioanaylzer and qPCR methods are used to qualify and quantify the sample library. Then the library products were used for sequencing.. Sequencing was performed on the Illumina HiSeq 4000 platform (Illumina company, San Diego, California, USA).