Reduced complexity ddRAD-seq for conifers

A total of 252 individuals were included in ddRAD-seq experiment [34 (link)]. Compared with the original restriction site-associated DNA sequencing (RAD-seq) [35 (link)], ddRAD-seq effectively reduced the complexity of studying the large genome of conifers [36 (link)]. The total genomic DNA (250 ng) from each sample was digested with SphI and PstI, ligated with Y-shaped adaptors, and amplified by PCR using KAPA HiFi polymerase (KAPA Biosystems, Woburn, MA, USA). After PCR amplification with adapter-specific primer pairs (Access Array Barcode Library for Illumina Sequencers; Fluidigm, San Francisco, CA, USA), an equal amount of DNA from each sample was mixed and size-selected using BluePippin agarose gel cassettes (Sage Science, Beverly, MA, USA). The library fragments (~450 bp) were retrieved, and the quality of the library was checked using an Agilent 2100 Bioanalyzer with a high-sensitivity DNA chip (Agilent Technologies, Waldbronn, Germany). The library was sequenced using the Illumina^® HiSeq X platform (Illumina, San Diego, CA, USA) to generate 150 bp long paired-end reads (see details in Supplementary Materials S1). The raw ddRAD-seq data were deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA012397.