Hi All
I've been killing myself trying to find info on dealing with sample cross-contamination in RNA-Seq - lots of warnings to take precautions as cross-contamination is a real issue, but nobody talking about having experienced the issue and what was done to correct for it. Here's what I'm doing and what we found:
My main questions are:
Thanks
Michael
I've been killing myself trying to find info on dealing with sample cross-contamination in RNA-Seq - lots of warnings to take precautions as cross-contamination is a real issue, but nobody talking about having experienced the issue and what was done to correct for it. Here's what I'm doing and what we found:
- Using TruSeq RNA prep kit with the 12 indexes that come with it
- multiplexing no more than 5 samples to a lane
- take the necessary precautions when ligating the indices to the cDNA samples
- doing full transcriptome analysis comparing single gene knock-out samples to WT, so a contamination issue of 1 in 1000 can have a significant effect. We have found quite a few samples with this level of contamination.
- several different tissues involved, and the contamination we're concerned with at this point is cross-tissue
- have a list of 5 highly expressed, ~very specific genes for each tissue against which we compare each sample to check for contamination
- we're almost certain that the cDNA prep step is the culprit as all the contaminating tissues were processed no more than 4 tubes away from the contaminated sample, on an 8 strip tube
My main questions are:
- in what environment are others prepping their cDNA for sequencing and are any major precautions being taken to eliminate cross-contamination?
- once data has been generated in which cross-contamination has been observed, are there any data manipulation fixes that anyone knows about or is this data simply garbage?
Thanks
Michael
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