Differentially gene expression multispecies
Entering edit mode
3 months ago

Hi friends, how are you? I need your help with my study project. I have RNAseq data for 3 species (6 replicates per species), species form a genus within bedbugs. I would like to analyze the differential expression of these species, however I have doubts if the answers I get are real or methodological biases. At the moment I am using all assemblies (18 = 6 per species) to map the expression and I have interesting results, however I don't know if it could be technical bias.

forward my results

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#build reference with all assemblies (n =18)

kallisto index -i reference ( all assemblies) 

#analysed by sample (pairend)

kallisto quant -i reference.idx -o output --rf-stranded -b 100 r1.fasta r2.fasta

## estimetes 
Trinity/util/abundance_estimates_to_matrix.pl \
 --est_method kallisto --gene_trans_map reference.fasta.gene_trans_map \
 --name_sample_by_basedir --cross_sample_norm TMM --out_prefix outdir \
sample1, sample2 ...sample18

Trinity/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix  gene.counts.matrix --method edgeR --output out --dispersion 0.1

Trinity/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix gene.TMM.EXPR.matrix --max_genes_clust 1000000 -P 1e-3 -C 4

Trinity/Analysis/DifferentialExpression/define_clusters_by_cutting_tree.pl -R / diffExpr.P1e-3_C4.matrix.RData --Ptree 60

What do you guys think will be next to trust us in my results or improve them?

Gene Expression Multiespecies RNAseq • 246 views
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do you know if each taxon was sequenced as a separate batch or all the samples were sequenced together? Samples of each taxon cluster together, this can be biologically meaningful but also could be due to a batch effect.

Entering edit mode

Hi, buddy, sorry for the delay. On sequencing they were sequenced in the same batch and the conditions were the same. My doubt is that because I don't have a genome or transcriptome as a reference, I may be obtaining "non-real" data about the expression. I chose to use all assemblies (n=18 (6 per species)) to obtain the reference and analyze against this "super reference". My collaborators are unsure about the results, however the methodology is consistent with "good practices".

I would like to be certain that I could continue with this study.


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