Question: Is it right to merge different trinity transcript assembly of same plant ?
0
gravatar for Mahendra Gaur
2.0 years ago by
Siksha O Anusandhan University (SOA University),Bhubaneswar,Odisha,India
Mahendra Gaur0 wrote:

Dear All,

I have rna-seq data of 3 ginger sample of which are from different condition.

I have assembled them using trinity tool, hence i got three different transcript assembly.

Since public database doesn't have reference genome of ginger, therefore to build own custom transcript genome of ginger i merged and cluster all three trinity assemblies following this workflow

Workflow:-

Merging all samples trinity assemblies

cat 0-trinity_out/Unigenes_S1.fasta 0-trinity_out/Unigenes_S3.fasta 0-trinity_out/Unigenes_S4.fasta > 0-trinity_out/Unigenes_all.fasta

Clustring merged assemlies at 100% identity

cd-hit-est -i 0-trinity_out/Unigenes_all.fasta -o 1-cdhit_out/cdhit_clstr.fasta -c 1.0 -n 8 -g 1 -r 1 -B 1 -p 1 &> 1-cdhit_out/cdhit_clstr.log

cat 1-cdhit_out/cdhit_clstr.fasta | python /home/blab/myscript/rename_multifasta.py Seq > 2-tgicl_out/cdhit_clstr.fasta

read_fasta -i 2-tgicl_out/cdhit_clstr.fasta | write_fasta -x -o 2-tgicl_out/cdhit_clstr_singleline.fasta

mv 2-tgicl_out/cdhit_clstr_singleline.fasta 2-tgicl_out/cdhit_clstr.fasta

Further clustring using TGICL

$TGICL -F cdhit_clstr.fasta -p 99 -l 50 -v 100 -c 3 -L -b blab:cbt@soa:mysql:127.0.0.1:tgicldb -R

cat asm_1/contigs asm_2/contigs asm_3/contigs asm_1/singlets asm_2/singlets asm_3/singlets > final_contigs.fasta

EvidentialGene::tr2aacds.pl pipeline script for processing final_contigs.fasta into the most biologically useful "best" set of mRNA, classified into "primary" and "alternate" transcripts.

/home/blab/Downloads/evigene/scripts/prot/tr2aacds_qsub.sh

The output is a neat pile of "okay" and "drop" transcripts

The "okay" set is annotated using trinotate tool to used as custom transcript genome.

ADD COMMENTlink written 2.0 years ago by Mahendra Gaur0

Combine the raw fastq files from each samples, and create a single trinity assembly. This will make downstream DGE analysis much easier, where you can specify conditions and replicates.

ADD REPLYlink written 2.0 years ago by st.ph.n2.4k
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