Testing for sex-specific variation at the Macquarie perch sex-linked region in other percichthyids using amplicon-based sequencing
To investigate whether the Macquarie perch sexing region has sex-linked variation in three other species of percichthyids, we amplified and sequenced ~400 bp encompassing the region, using primers conserved across three percichthyid genomes: Macquarie perch, golden perch and Murray cod (primers F2 and R3, Table C1). Amplicon sequencing was performed on an Illumina ISeq100 at GeneSEQ Sdn Bhd (Malaysia) using the unidirectional read configuration of 1 x 300 bp (details in Appendix A). Two males and two females of each of the four species were sequenced, along with two coastal Macquarie perch individuals of unknown sex but appearing to be one of each sex based on the PCR-RFLP sexing assay (list of samples in Supplementary Material S1). Reads in the fastq files were trimmed of poly-G sequences (a sequencing artefact in the newer NextSeq500/NovaSEQ6000/ISeq100 Illumina system) usingcutadapt (Martin, 2011) via the option –nextseq-trim . Each individual was then genotyped for the 400-bp amplicon region using containerized best-practice GATK v4.1.0 protocol (Van der Auwera et al., 2013), available on the docker repository under tag:trust1/gatk:version0.3.1 . The ~600 bp region used for primer design was also used as a reference (LKNJ01002546 bases 82756- 83361 for Murray cod and trout cod, VMKM01003747 bases 16365-16988 for golden perch and scaffold 633 bases 93001-93600 for Macquarie perch).