Dear all,
I have recently sequenced RNA populations from two different cell types that were separated using FACS (negatives and positives). The aim of the experiment is to identify Deferentially expressed genes (DEGs) in the positive cell types. Although I have three biological replicates for each of these conditions, two of my replicates for the positive condition appear to be nearly identical: please see the attached images (TP1 and TP3 overlap in the PCA). I have made sure that I did not mix up reads from these two replicates and that they were two different samples (i.e., it wasn't a technical replicate but a true biological replicate).
So, I wonder..
1) what are the factors that could result in such a scenario, besides the explanation that these two samples had more or less the same expression level - although, I am not sure how frequently this could happen.
2) What effect can this have on my analyses? I guess, it would decrease the power of detecting DEGs.
3) If so, can I rely on the list of DEGs already identified by this approach?
3) Finally, what would you recommend:
a) re-sequencing one of the positive replicates - this would be a major setback as getting this done in the first place was a hassle; or
b) omit one of these identical replicates, and redo the analyses? Although it would be the same as analyzing without omitting one of the replicates, I guess.
Thank you very much in advance for your time and advise,
Best Regards,
Kartik
I have recently sequenced RNA populations from two different cell types that were separated using FACS (negatives and positives). The aim of the experiment is to identify Deferentially expressed genes (DEGs) in the positive cell types. Although I have three biological replicates for each of these conditions, two of my replicates for the positive condition appear to be nearly identical: please see the attached images (TP1 and TP3 overlap in the PCA). I have made sure that I did not mix up reads from these two replicates and that they were two different samples (i.e., it wasn't a technical replicate but a true biological replicate).
So, I wonder..
1) what are the factors that could result in such a scenario, besides the explanation that these two samples had more or less the same expression level - although, I am not sure how frequently this could happen.
2) What effect can this have on my analyses? I guess, it would decrease the power of detecting DEGs.
3) If so, can I rely on the list of DEGs already identified by this approach?
3) Finally, what would you recommend:
a) re-sequencing one of the positive replicates - this would be a major setback as getting this done in the first place was a hassle; or
b) omit one of these identical replicates, and redo the analyses? Although it would be the same as analyzing without omitting one of the replicates, I guess.
Thank you very much in advance for your time and advise,
Best Regards,
Kartik