Amplicon sequencing of X. hellerii x (X. maculatus x X. hellerii) hybrids: muscle
Fin clip, or muscular tissue, was digested by Protease K at room temperature for 1 hr. The lysate was then transferred to 2.0 ml collection tubes. DNA isolation was performed by a QIAcube HT (Qiagen) automated bio-sample isolation system, with reagent contained in QIAamp 96 DNA QIAcube HT Kit. The isolation system is equipped with a robotic arm with 8 pipettes. Each pipette is able to pick and eject pipette tips, self-clean, and transfer liquids between wells/columns, or between master reservoirs and wells/columns in standard 96-well plate formats. Each sample was independently maintained throughout the isolation process. Concentrations of DNA samples were measured using Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA), and adjusted for sequencing library preparation. A total of 2,017 probe sets were produced for amplicon sequencing. Amplicons were custom made using Illumina Genotype Ne library preparation kit, with i7 and i5 indices incorporated into adaptor sequences added to each end of PCR products amplified by capture probes. Pooled sequencing libraries were sequenced on Illumina MiSeq platform employing a 75 bp paired-end sequencing strategy (Illumina, San Diego, California).