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statonlab / hardwoods_site

Hardwoods Genomics bugs, data loading, and general issues
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Data loading: Ginkgo biloba #241

Open bradfordcondon opened 6 years ago

bradfordcondon commented 6 years ago

https://www.researchgate.net/publication/299471846_Global_comparative_analysis_of_expressed_genes_in_ovules_and_leaves_of_Ginkgo_biloba_L

mestato commented 6 years ago

SRA: http://www.ncbi.nlm.nih.gov/Traces/sra

GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64502

Bioproject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA271136

patricksis commented 5 years ago

Using two SRR files: SRR1734725 - fully expanded leaves

of bases = 568.3M

    size = 367.8Mb

SRR1734724 - Mature ovules

of bases = 610.5M

    size = 392.8Mb
patricksis commented 5 years ago

fastqc:

for f in ../raw_reads/*.fastq
do
    filename=$(basename "$f")
    base="${filename%%.fastq*}"
    echo "filename $filename base $base"
    mkdir $base.fastQC

    /staton/software/FastQC-v0.11.5/FastQC/fastqc -o $base.fastQC $f >& $base.fastQC.out &
done
patricksis commented 5 years ago

trimmomatic:

for f in ../raw_reads/*.fastq
do
 BASE=$( basename $f | sed 's/.fastq*//g')
 echo "f $f"
 echo "BASE $BASE"

 module load java/jdk8u181

 java -jar /staton/software/Trimmomatic-0.38/trimmomatic-0.38.jar SE -phred33 $f $BASE.trim.fastq ILLUMINACLIP:/staton/software/Trimmomatic-0.38/adapters/all.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
done
patricksis commented 5 years ago

RCorrector:

perl /staton/software/rcorrector/run_rcorrector.pl -s /staton/projects/undergrads/ginkgo_biloba/2_trimmomatic/SRR1734725_1.trim.fastq,/staton/projects/undergrads/ginkgo_biloba/2_trimmomatic/SRR1734724_1.trim.fastq -od /staton/projects/undergrads/ginkgo_biloba/3_RCorrector
patricksis commented 5 years ago

trinity:

export PATH=$PATH:/staton/software/bowtie2-2.2.7/

module load java/jdk8u181

/staton/software/trinityrnaseq-Trinity-v2.4.0/Trinity --seqType fq --single /staton/projects/undergrads/ginkgo_biloba/3_RCorrector/SRR1734724_1.trim.cor.fq,/staton/projects/undergrads/ginkgo_biloba/3_RCorrector/SRR1734725_1.trim.cor.fq --CPU 6 --max_memory 20G --output se_trinity_out_dir
patricksis commented 5 years ago

Skewer:

for R in /staton/projects/undergrads/ginkgo_biloba/raw_reads/*.fastq
do
 BASE=$( basename $R | sed 's/_1.fastq*//g')
 echo "R $R"
 echo "BASE $BASE"

 /staton/software/skewer/skewer \
 -x /staton/software/Trimmomatic-0.38/adapters/all.fa \
 -l 30 \
 $R \
 -o $BASE \
 >& $BASE.trim_output &

done
patricksis commented 5 years ago

indexing:

/staton/software/STAR-2.6.1a/bin/Linux_x86_64/STAR \
 --runMode genomeGenerate \
 --genomeDir genomeDir \
 --genomeFastaFiles ../4_Trinity/se_trinity_out_dir/Trinity.fasta &
patricksis commented 5 years ago

Alignment:

for f in ../5_skewer/*.log
do
 BASE=$( basename $f | sed 's/.log*//g')
 echo "BASE $BASE"

 /staton/software/STAR-2.6.1a/STAR \
  --genomeDir genomeDir \
  --readFilesIn ../5_skewer/$BASE.fastq \
  --outSAMtype BAM SortedByCoordinate \
  --outFileNamePrefix $BASE. &

done
patricksis commented 5 years ago

HTseq:

for bam in ../6_star/*.bam
do
 base=$( basename $bam | sed 's/.sorted.bam//g')

 echo "bam $bam"
 echo "base $base"
 echo "--"

 /staton/software/htseq-count \
  --format=bam \
  --order=pos \
  --stranded=no \
  --type=gene \
  --idattr=ID \
  $bam \
  ../4_Trinity/se_trinity_out_dir/Trinity.fasta.gff3 \
  >$base.counts.txt \
  2> $base.out &

echo "-------"

done
patricksis commented 5 years ago

blast refinement:

/staton/software/ncbi-blast-2.7.1+/bin/blastp \
 -query Trinity.fasta.transdecoder_dir/longest_orfs.pep \
 -db /staton/libraries/uniprot/uniprot_sprot.fasta \
 -max_target_seqs 1 \
 -outfmt 6 \
 -evalue 1e-5 \
 -num_threads 10 > blastp.outfmt6
patricksis commented 5 years ago

TransDecoder2:

/staton/software/TransDecoder-3.0.0/TransDecoder.Predict -t ../4_Trinity/se_trinity_out_dir/Trinity.fasta --retain_blastp_hits blastp.outfmt6 &
patricksis commented 5 years ago

Transcriptome assembled, no issues so far!

patricksis commented 5 years ago

Organism Page: https://www.hardwoodgenomics.org/organism/Ginkgo/biloba Publication: https://www.hardwoodgenomics.org/Publication/3732396 Transcriptome assembly: https://www.hardwoodgenomics.org/Transcriptome-assembly/3732397?tripal_pane=group_summary_tripalpane InterProScan annotation: https://www.hardwoodgenomics.org/InterProScan-annotation/3732398 Swissprot annotation: https://www.hardwoodgenomics.org/BLAST-annotation/3732399 trembl annotation: https://www.hardwoodgenomics.org/BLAST-annotation/3732400 CDS fasta loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/828387 Peptide fasta loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/828388 Publish tripal content: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/828769 Trembl results loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/831432 Swissprot results loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/831436 IPS results loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/831440

patricksis commented 5 years ago

Swissprot:

#PBS -N swissprot_BLAST
#PBS -S /bin/bash
#PBS -j oe
#PBS -A ACF-UTK0011
#PBS -t 1-200
#PBS -l nodes=1:ppn=2
#PBS -l walltime=04:00:00

cd $PBS_O_WORKDIR

module load blast

blastx \
 -query /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/raw_data/BLAST_split/Trinity.fasta.fixed.cds.$PBS_ARRAYID \
 -db /lustre/haven/gamma/staton/library/uniprot/uniprot_sprot.fasta \
 -out /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/BLAST/swissprot/g_biloba.$PBS_ARRAYID.xml \
 -evalue 1e-5 \
 -outfmt 5
patricksis commented 5 years ago

trembl:


#PBS -N trembl_BLAST
#PBS -S /bin/bash
#PBS -j oe
#PBS -A ACF-UTK0011
#PBS -t 1-200
#PBS -l nodes=1:ppn=2
#PBS -l walltime=12:00:00

cd $PBS_O_WORKDIR

module load blast

blastx \
 -query /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/raw_data/BLAST_split/Trinity.fasta.fixed.cds.$PBS_ARRAYID \
 -db /lustre/haven/gamma/staton/library/uniprot/uniprot_trembl_plants_July_2018.fasta \
 -out /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/BLAST/trembl/g_biloba_trembl.$PBS_ARRAYID.xml \
 -evalue 1e-5 \
 -outfmt 5
patricksis commented 5 years ago

IPS:

#PBS -N g_biloba_ips
#PBS -A ACF-UTK0011
#PBS -S /bin/bash
#PBS -t 1-200
#PBS -j oe
#PBS -l nodes=1:ppn=4
#PBS -l walltime=6:30:00

cd $PBS_O_WORKDIR

module load python3

/lustre/haven/gamma/staton/software/interproscan-5.34-73.0/interproscan.sh \
 -i /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/raw_data/IPS_split/Trinity_noAst.pep.$PBS_ARRAYID \
 -f XML \
 -d /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/IPS/xmls \
 --disable-precalc \
 --iprlookup \
 --goterms \
 --pathways \
 --tempdir /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/IPS/TMP \
 > /lustre/haven/gamma/staton/projects/undergrads/ginkgo_biloba/IPS/xmls/TMP/$PBS_ARRAYID.out
patricksis commented 5 years ago

bdb: transcrips: https://www.hardwoodgenomics.org/content/ginkgo-biloba-transcrips peptides: https://www.hardwoodgenomics.org/content/ginkgo-biloba-peptides

patricksis commented 5 years ago

KEGG annotation: https://www.hardwoodgenomics.org/KEGGresults/3890870?tripal_pane=gp_sio__annotation47730 KEGG loader: https://www.hardwoodgenomics.org/admin/tripal/tripal_jobs/view/836053