##CD-HIT redundancy removal for each assembly (k25, k29, k32)
cd-hit-est -i Dunnart_trinity_k25_output.Trinity.fasta -c 1 -M 0 -T 0

##Run RSEM-EVAL on each assembly (k25, k29, k32)
./rsem-eval/rsem-eval-calculate-score --transcript-length-parameters parameter_file -p 10 --time --bowtie2 --paired-end fastq_list.txt Dunnart_trinity_k25_output.Trinity.fasta ./output/rsem_eval_1 142 

##Compare RSEM-EVAL scores
cat output/rsem_eval_1.score | awk '$1 == "Score"'
cat output/rsem_eval_2.score | awk '$1 == "Score"'
cat output/rsem_eval_3.score | awk '$1 == "Score"'

##rnaQUAST
rnaQUAST.py --threads 10 --transcripts Dunnart_trinity_k25_output.Trinity.fasta Dunnart_trinity_k29_output.Trinity.fasta Dunnart_trinity_k32_output.Trinity.fasta ~/rnaQUAST__output_mammalia_BUSCO --disable_infer_genes --disable_infer_transcripts --busco

##Identify all long ORFs
TransDecoder.LongOrfs -t Dunnart_trinity_k29_output.Trinity.fasta

##Predict which ORFs are likely to be coding
TransDecoder.Predict --cpu 10 -t Dunnart_trinity_k29_output.Trinity.fasta

##Capture likely homolog information (combined protein db: human, mouse, Tasmanian devil, koala, tammar wallaby, gray short-tailed opossum, and numbat)
blastx -query Dunnart_trinity_k29_output.Trinity.fasta -db uniprot_combined_species_filt.fasta -max_target_seqs 1 -outfmt 6 -evalue 1e-5 -num_threads 10 > sprot.blastx.outfmt6

##Searching the predicted protein sequences
blastp -query Dunnart_trinity_k29_output.Trinity.fasta.transdecoder.pep -db uniprot_combined_species_filt.fasta -max_target_seqs 1 -outfmt 6 -evalue 1e-5 -num_threads 10 > sprot.blastp.outfmt6

##Load Trinotate.sqlite database
$TRINOTATE_HOME/Trinotate ./Trinity/Trinotate/Sequence_databases/Trinotate.sqlite init --gene_trans_map Dunnart_trinity_k29_output.Trinity.fasta.gene_trans_map --transcript_fasta Dunnart_trinity_k29_output.Trinity.fasta --transdecoder_pep Dunnart_trinity_k29_output.Trinity.fasta.transdecoder.pep

##HMMER search against the Pfam database
hmmscan --cpu 12 --domtblout TrinotatePFAM.out ../data/trinotate_data/Pfam-A.hmm Dunnart_trinity_k29_output.Trinity.fasta.transdecoder.pep

##Predict signal peptides
signalp -fasta Dunnart_trinity_k29_output.Trinity.fasta.transdecoder.pep -stdout signalp.out

##Predict transmembrane helices
tmhmm --short < Dunnart_trinity_k29_output.Trinity.fasta.transdecoder.pep > tmhmm.out

##Compile all predictions
$TRINOTATE_HOME/Trinotate Trinotate.sqlite LOAD_swissprot_blastx sprot.blastx.outfmt6
$TRINOTATE_HOME/Trinotate Trinotate.sqlite LOAD_swissprot_blastp sprot.blastp.outfmt6
$TRINOTATE_HOME/Trinotate Trinotate.sqlite LOAD_pfam TrinotatePFAM.out
$TRINOTATE_HOME/Trinotate Trinotate.sqlite LOAD_signalp signalp.out
$TRINOTATE_HOME/Trinotate Trinotate.sqlite LOAD_tmhmm tmhmm.out