I tried to use trinity to assemble my RNA-seq raw data and find some differential expression genes between control and experiment groups. Two RNA-seq samples without biological replicates were contained in my analyses, one is control and another is experiment. As a newer of NGS analyses, I followed the protocol of trinity website (http://trinityrnaseq.github.io/analysis/diff_expression_analysis.html). However, only 40 DE genes were identified and our target upregulation gene was not contained in these genes. I pasted my command here and ask for you help me to find some unsuitable treatment. Thanks a lot!
Note: I have two samples and four fastq files
$ align_and_estimate_abundance.pl --transcripts Trinity.fasta --seqType fq --left sample1_1.fastq --right sample1_2.fastq --est_method RSEM --aln_method bowtie --SS_lib_type RF --thread_count 24 --trinity_mode --prep_reference # Align reads to reference through bowtie software;
After this step, we obtained two isoforms.results and two gene.results files, we used genes.results file in further analyses which named as sample1.genes.results and sample2.genes.results
$ abundance_estimates_to_matrix.pl --est_method RSEM sample1.genes.results sample2.genes.results
$ run_DE_analysis.pl --matrix matrix.count.matrix -method edgeR
$ cd edgeR ...dir/
$ analyze_diff-expr.pl --matrix /home/.../Trinity_trans.TMM.fpkm.matrix -P 1e-3 -C 2