Hi all,

I'm performing an experiment using RNA-seq data which will subsequently involve Differential Gene Analysis, and I'm experiencing the following problem. I'm using the trinity rna-seq suite, I've used kallisto as well as RSEM to check the replicate correlation. The species is crocodilian in nature. (no reference genome yet for my species)

1. On comparing replicates for the same tissue, eg. Brain or genital ridge, I'm observing that correlation between replicates for Brain sample are not correlating together. (quantitative heatmap)

   sample 1 -> cond 1 
   sample 2 -> cond 1
   sample 3 -> cond 2 
   sample 4 -> cond x (sample 4 should segregate with cond 2)
   

   Infact, sample 4 doesn't have a correlation (very less or negative) with other Brain tissue.

2. On adding more samples from other tissues, the outlier changes entirely. And I'm observing that kidney samples or skin samples correlate with other brain samples (any tissue correlates somewhat well with other brain samples) barring sample 3 (in example above.)

Could someone point out what might be the probable reason for this behavior, other than sequencing and sample preparation error?

Harish