##########Prepare directories
mkdir SI006_AglaiaFungus
cd SI006_AglaiaFungus
mkdir RawFastq
cd RawFastq

##########Download data
wget -r -nH --cut-dirs=4 ftp:/path/to/directory/*.gz

##########Prepare directories
cd /datanfs12/SI006_AglaiaFungus
mkdir Alignment
cd Alignment


##########Removing adaptor sequences from library, then splitting by barcode
nohup zcat /datanfs12/SI006_AglaiaFungus/RawFastq/SISI006B_S6_L002_R1_001.fastq.gz | fastq_illumina_filter --keep N -v | fastx_clipper -Q33 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -n -v -o stem_fungus_R1_clipped.fastq &
nohup zcat /datanfs12/SI006_AglaiaFungus/RawFastq/SISI006B_S6_L002_R2_001.fastq.gz | fastq_illumina_filter --keep N -v | fastx_clipper -Q33 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -n -v -o stem_fungus_R2_clipped.fastq &


##########Prepare directories
cd /datanfs12/SI006_AglaiaFungus
mkdir Alignment
cd Alignment
mkdir stem_fungus
cd /datanfs12/SI006_AglaiaFungus/Alignment/stem_fungus

###########Run trinity
/usr/local/bin/trinityrnaseq-Trinity-v2.8.4/Trinity --SS_lib_type RF --seqType fq --left ../stem_fungus_R1_clipped.fastq --right ../stem_fungus_R2_clipped.fastq --CPU 4 --max_memory 5G --output trinity_stem_fungus --full_cleanup &

###########Prepare database
/home/iwasaki/Trinotate-Trinotate-v3.1.1/admin/Build_Trinotate_Boilerplate_SQLite_db.pl  Trinotate
makeblastdb -in uniprot_sprot.pep -dbtype prot
gunzip Pfam-A.hmm.gz
hmmpress Pfam-A.hmm

###########Run transdecorder
TransDecoder.LongOrfs -S -t /datanfs12/SI006_AglaiaFungus/Alignment/stem_fungus/trinity_stem_fungus.Trinity.fasta &
TransDecoder.Predict -t /datanfs12/SI006_AglaiaFungus/Alignment/stem_fungus/trinity_stem_fungus.Trinity.fasta &


###########Run blast+
blastx -query /datanfs12/SI006_AglaiaFungus/Alignment/stem_fungus/trinity_stem_fungus.Trinity.fasta -db uniprot_sprot.pep -num_threads 4 -max_target_seqs 1 -outfmt 6 -evalue 1e-3 > blastx.outfmt6 &
blastp -query /datanfs12/SI006_AglaiaFungus/Alignment/stem_fungus/trinity_stem_fungus.Trinity.fasta.transdecoder.pep -db uniprot_sprot.pep -num_threads 4 -max_target_seqs 1 -outfmt 6 -evalue 1e-3 > blastp.outfmt6 &


grep IF4A blastx.outfmt6

###########Run hmmer
hmmscan --cpu 4 --domtblout TrinotatePFAM.out Pfam-A.hmm trinity_stem_fungus.Trinity.fasta.transdecoder.pep > pfam.log &



###########Run signalP
#split stem_fungus/trinity_stem_fungus.Trinity.fasta.transdecoder.pep into 8 files

signalp -f short -n signalp_1.out trinity_stem_fungus.Trinity.fasta.transdecoder_split1.pep &
signalp -f short -n signalp_2.out trinity_stem_fungus.Trinity.fasta.transdecoder_split2.pep &
signalp -f short -n signalp_3.out trinity_stem_fungus.Trinity.fasta.transdecoder_split3.pep &
signalp -f short -n signalp_4.out trinity_stem_fungus.Trinity.fasta.transdecoder_split4.pep &
signalp -f short -n signalp_5.out trinity_stem_fungus.Trinity.fasta.transdecoder_split5.pep &
signalp -f short -n signalp_6.out trinity_stem_fungus.Trinity.fasta.transdecoder_split6.pep &
signalp -f short -n signalp_7.out trinity_stem_fungus.Trinity.fasta.transdecoder_split7.pep &
signalp -f short -n signalp_8.out trinity_stem_fungus.Trinity.fasta.transdecoder_split8.pep &

cat signalp_* > signalp.out


###########Run tmhmm
tmhmm --short < trinity_stem_fungus.Trinity.fasta.transdecoder.pep > tmhmm.out &

###########Run rnammer
/home/iwasaki/Trinotate-Trinotate-v3.1.1/util/rnammer_support/RnammerTranscriptome.pl --transcriptome trinity_stem_fungus.Trinity.fasta --path_to_rnammer /home/iwasaki/rnammer &

###########Sqlite database
#Initial import of transcriptome and protein data
Trinotate Trinotate.sqlite init --gene_trans_map trinity_stem_fungus.Trinity.fasta.gene_trans_map --transcript_fasta trinity_stem_fungus.Trinity.fasta --transdecoder_pep trinity_stem_fungus.Trinity.fasta.transdecoder.pep

###########Transdecoder protein search results:
Trinotate Trinotate.sqlite LOAD_swissprot_blastp blastp.outfmt6
Trinotate Trinotate.sqlite LOAD_pfam TrinotatePFAM.out
Trinotate Trinotate.sqlite LOAD_tmhmm tmhmm.out
Trinotate Trinotate.sqlite LOAD_signalp signalp.out

###########Trinity transcript search results
Trinotate Trinotate.sqlite LOAD_swissprot_blastx blastx.outfmt6
Trinotate Trinotate.sqlite LOAD_rnammer trinity_stem_fungus.Trinity.fasta.rnammer.gff

###########Report generation:
Trinotate Trinotate.sqlite report --incl_pep --incl_trans> stem_fungus_trinotate_annotation_report.xls
