Background
We have an RNA-seq project on small intestine. The experimental design were like this: Four physiological states, each with three individuals (biological replicates), each individual with two samples (two parts of small intestine), thus 4 physiological states × three individuals × two parts. The reason we included two parts of same individual’s small intestines is that we want to take consideration of tissue heterogeneity, by pre-analysis of these replicates the difference between two parts is actually not significant.
Question
Here is my question, when I applied a generalized linear model for the differential expression analysis on samples from two physiological states (e.g. DESeq2), should I treat samples as independent one thus 6 vs 6, or merge two samples from same individuals thus 3 vs 3. For the later one, can I regard it as technical replicate and merge reads from the two part together, or merge counts calculated from the two parts (e.g. collapseReplicates function in DESeq2). Or, should I include "individuals" as a blocking factor in the differential expression analysis.
There is an intensive discussion about biological replicates and technical replicates, like these https://www.biostars.org/p/54380/
https://www.biostars.org/p/305087/
https://www.biostars.org/p/304008/#
but I am still confused about my situation, should I treat the two tissue parts from same individuals as biological or technical replicates.
Two parts of small intestine from same individuals, It's technical or biological replicates?
Hi Haijian
I would like to call “two parts of the small intestine from the same individual” as technical replicates rather than biological replicates! Cause two samples were from the same individual. However, hope these two parts of the small intestine are located close enough, otherwise this so called technical repeat may not be perfect enough to generate similar results due to the heterogeneity of small intestine .
Thanks Pro. Pan
It is a ticklish question to distinguish between technical and biological replicate in our situation, thank you for your time and your answer. The work the question from have been peer-reviewed and published (see details below). I’d like to share our answers. Firstly, we tried to answer two question, one is whether the variation between the two replicate is from biological factors (e.g. sex, age, or tissue heterogeneity), or from technical factors (e.g. sequencing lanes, library generation). It is reasonable that the two parts of small intestine were not biological replicates, just as you said, two samples were from the same individual and they shared same genetic background, then the variation between the replicates were from sequencing or other technical factors. However, as our original intention is inducing the variation from tissue heterogeneity, although they come from close part of small intestine, it’s not precise to just regard them as technical replicates and merge them in downstream analysis. The other question we want to answer is, how to take consideration of tissue heterogeneity but also avoid pseudoreplication caused by the same origian of the replicates. In our work, we randomly assign the two samples from the same individual to “PartA” and “PartB” and incorporate the blocking design into differential expression tools with ~sampled part + physiological states. By fitting design model above, we can compare the four physiological states with adjustment of the baseline differences from tissue heterogeneity (see details in Article Gut transcriptomic changes during hibernation in the greater...
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