title: Illumina HiSeq 4000 sequencing of SAMD00469673
library_construction_protocol: "Before DNA extraction, a piece of adductor muscle was cut from each sample of the unionid mussels. Total DNA was isolated from specimens of mussels using a NucleoSpin Tissue kit (TaKaRa, Shiga Prefecture, Japan), according to the manufacturer's instructions. We performed double digest restriction site-associated DNA library (ddRAD) sequencing on 87 genomic DNA samples (Table S1) using the procedure reported by Peterson et al. (2012) with slight modifications. RNase A was added to the DNA extracted from each specimen. Approximately 40 ng of DNA was digested using two restriction enzymes (EcoRI and MspI). P1 and P2 adaptors, reported by Peterson et al. (2012), were ligated to DNA fragments from each sample. The ligated samples were multiplexed and purified using a NucleoSpin gDNA Clean-up kit (Macherey-Nagel). A size selection of DNA fragments between 300?350 bp was performed using Pippin prep (Sage Science, MA, USA). The DNA fragment was amplified by polymerase chain reaction (PCR) and purified. The constructed DNA libraries were sent to the University of Oregon (USA) and sequenced using Illumina Hi-seq 4000 single-end sequencing. Raw sequence data were analyzed using pyRAD (Eaton, 2014) and ipyrad (Eaton and Overcast, 2020). Identical settings were used in each program, which resulted in similar output files of different formats."