Question: Low DE gene numbers after Trinity analysis
gravatar for Ginsea Chen
3.7 years ago by
Ginsea Chen120
Chinese Academy of Tropical Agricultural Sciences, Danzhou, China
Ginsea Chen120 wrote:

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 ( 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

$ --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

$ --est_method RSEM  sample1.genes.results sample2.genes.results

$ --matrix matrix.count.matrix -method edgeR

$ cd edgeR ...dir/

$ --matrix /home/.../Trinity_trans.TMM.fpkm.matrix -P 1e-3 -C 2


dge rna-seq trinity • 1.1k views
ADD COMMENTlink modified 3.7 years ago by h.mon24k • written 3.7 years ago by Ginsea Chen120

Why does it surprise you, that DE analyses without replication yield a low number of significant genes? Sometimes I think that succumbing to the pressure from 'experimentalists' to implement magic tricks for providing some sort of 'p-values' out of non-replicated experiments was a really bad idea, top candidate for What Are The Most Common Stupid Mistakes In Bioinformatics?

ADD REPLYlink written 3.7 years ago by Michael Dondrup46k
gravatar for h.mon
3.7 years ago by
h.mon24k wrote:

I like this paper: RNA-seq differential expression studies: more sequence or more replication?

You could also try Biostar's "Live search" using "without replicates", there will be plenty reading.

ADD COMMENTlink written 3.7 years ago by h.mon24k
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