Dear all!

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