Assembly, Annotation, Quantification, and Differential Expression Analysis of Shorea sp. Transcriptome
Ahmad Husaini AHS Suhaimi
Abstract
Assembly, annotation, and quantification of transcripts from RNA-seq reads of Shorea sp. transcriptome followed by differential expression analysis using open source tools.
List of tools:
Trinity (Home · trinityrnaseq/trinityrnaseq Wiki · GitHub)
TransDecoder (Home · TransDecoder/TransDecoder Wiki · GitHub)
NCBI Blast suite (Download BLAST Software and Databases Documentation (nih.gov))
InterProScan (Download - InterPro (ebi.ac.uk))
CDHIT suite (CD-HIT Official Website (ucsd.edu))
Salmon (Salmon - Salmon 1.6.0 documentation)
DESeq2 (Bioconductor - DESeq2)
Steps
Transcript Assembly
Obtain the assembly statistics
$TRINITY_HOME/util/TrinityStats.pl Trinity.fasta > Assembly_stats.txt
Assemble the reads using Trinity assembler
Trinity --seqType fq --max_memory 245G --left reads.PAPER1.left.fq --right reads.PAPER1.right.fq --CPU 20 --trimmomatic --monitoring > runPAPER1.log 2>&1 &
Removal of Transcript Redundancy
Extract the longest open reading frame (ORF) using TransDecoder
TransDecoder.LongOrfs -t Trinity.fasta
Query the extracted ORFs against Uniprot database using blastp
blastp -query transdecoder_dir/longest_orfs.pep -db uniprot_sprot.fasta -max_target_seqs 1 -outfmt 6 \
-evalue 1e-5 -num_threads 10 > blastp.outfmt6
Predict the single best ORF for each transcript
TransDecoder.Predict -t Trinity.fasta --single_best_only --retain_blastp_hits blastp.outfmt6
Remove highly similar protein sequences using CD-HIT
cd-hit -i Trinity_paper1.pep.fasta -o Trinity_paper1_output -T 0 -M 0 -d 0 -c 1 -n 5
Transcript Annotation
Query the nonredundant nucleotide sequences against A. thaliana proteome
blastx -query Trinity_reduced_output.fasta -db Araport11_genes.201606.pep.fasta -evalue 1e-10 -num_threads 33 -max_taget_seqs 1 -outfmt 6 -out blastx_trasncript_aradb_e10.outfmt6
Query the nonredundant protein sequences against Pfam, PANTHER, GO, and KEGG databases
./interproscan.sh -i Trinity_reduced_output.pep.fasta -t n -appl Pfam,PANTHER -goterms -iprlookup -pa --cpu 30
Transcript Quantification
Align and estimate transcript abundance using Salmon
$TRINITY_HOME/util/align_and_estimate_abundance.pl --transcript Trinity.fasta --seqType fq --samples_file samples_description.txt --est_method salmon --aln_method bowtie2 --thread_count 33 --prep_reference --trinity_mode --coordsort_bam
Construct matrices of counts and normalized values
$TRINITY_HOME/util/abundance_estimates_to_matrix.pl --est_method salmon --out_prefix Paper1_comORF_cdhit_only_c1 --name_sample_by_basedir leaf_1312_rep1/quant.sf leaf_1312_rep2/quant.sf leaf_1404_rep1/quant.sf leaf_1404_rep2/quant.sf leaf_1406_rep1/quant.sf leaf_1406_rep2/quant.sf --gene_trans_map Paper1_comORF_cdhit_only_c1.fasta.gene_trans_map
Differential Expression Analysis
Perform pairwise comparison between the time points
$TRINITY_HOME/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix quant.sf/gene.counts.matrix --method DESeq2 --samples_file samples_description_DESeq.txt --contrasts contrast_DESeq.txt
Extract the differentially expressed transcripts at log2 fold change ≥ 1, FDR < 0.05
$TRINITY_HOME/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix ../ComORF_cdhit_only_c1.isoform.TMM.EXPR.matrix -C 1 -P 0.05 --samples ../samples_description.txt
