Transcriptome assembly
Transcriptome assembly was conducted on the University of Sydney High
Performance Computer, Artemis. FastQC v0.11.8 (Andrews, 2010) was used
to assess the quality of raw reads. Trimmomatic v0.39 (Bolger et al.,
2014) was used to quality trim reads specifying TruSeq3-PE adapters,
SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5 and MINLEN:25. The
repeat-masked genome was indexed and reads aligned with HiSat2 v2.1.0
(Kim et al., 2019). SamTools v1.9 view and sort converted the files to
coordinate-sorted BAM format. A GTF for each transcriptome was generated
with StringTie v2.1.6 (Pertea et al., 2015). Aligned RNAseq reads were
merged into transcripts and filtered to remove transcripts found in only
one tissue with fragments per kilobase of transcript per million mapped
fragments (FPKM) < 0.1, using TAMA-merge v2020/12/17 (Kuo et
al., 2020) and CPC2 v2019-11-19 (Kang et al., 2017). TransDecoder v2.0.1
(Haas, 2022) predicted open reading frames in the resulting global
transcriptome. The completeness of the global transcriptome was assessed
using BUSCO v5.2.2 in ‘transcriptome’ mode with the Aves_odb10 lineage
on Galaxy Australia.