Low DE gene numbers after Trinity analysis
Entering edit mode
6.3 years ago
Ginsea Chen ▴ 130

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


RNA-Seq Trinity DGE • 1.6k views
Entering edit mode

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?

Entering edit mode
6.3 years ago
h.mon 33k

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.


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