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).