Illumina NextSeq 500 single-read sequencing of SAMD00506565
Genomic DNA was digested with the SbfI restriction enzyme (SbfI-HF; New England Biolabs). A modified Solexa P1 adapter (Illumina) containing six bases of index sequence was ligated to the cleaved DNA with T4 DNA Ligase (New England Biolabs). The P1-ligated fragments from each specimen were pooled and sheared by sonication with the focal size of 300 bp. Fragments ranging from 250 bp to 600 bp were recovered from 1.8% agarose gel, and their termini were repaired with Mighty Cloning Reagent Set-Blunt End (Takara). Exo-minus Klenow DNA Polymerase (Epicentre) produced 3'-adenine-protruding ends of the repaired fragments. A modified Solexa P2 adapter (Illumina) was ligated to the fragments. With the P5 and P7 PCR primers (Illumina), the P1- and P2-ligated fragments were subjected to 12 cycles of PCR (Phusion High-Fidelity DNA Polymerase; New England Biolabs) in eight separate tubes. The post-PCR mixtures were combined and purified with AMPure XP beads (Beckman Coulter), and the resulting DNA pool was referred to as a RAD library. 