Question: Single sample on flow cell with less than half as many reads as others on the same cell
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gravatar for mchair18
18 days ago by
mchair180
United States
mchair180 wrote:

Title pretty much says it all, but recently received results back from my core facility for a ChIP-Seq run, and one sample in particular had less than half as many reads identified as the rest of the samples. What would cause this? I would think that any issue with identifying the index itself would apply across all samples, so my mind initially moves to the possibility that less of this sample was loaded on the flow cell versus the others. Any ideas are much appreciated!

ADD COMMENTlink modified 18 days ago • written 18 days ago by mchair180

Maybe there is issue with library pooling or the quantification done before pooling.

ADD REPLYlink written 18 days ago by arup1.7k

Did you pool the samples yourself or did the core do it? If you did it, how did you quantify each sample?

ADD REPLYlink written 18 days ago by ATpoint23k

My initial thought was that there may have been an issue with quantification but I wanted to see if there were some other possibilities before I went back to the core with my concerns.

We did not pool or quantify any of the samples ourselves, each of these steps were performed by our core facility.

ADD REPLYlink written 18 days ago by mchair180

Use Add Reply please. If they did it you can always contact them and ask for clarification. I guess uneven loading is the most likely explanation.

ADD REPLYlink written 18 days ago by ATpoint23k

My apologies. Thank you for the input!

ADD REPLYlink written 18 days ago by mchair180

If your facility does not do proper quantification then this is not surprising. Keep in mind that libraries with smaller inserts preferentially cluster. Yes this does mean that if you have adapter dimer contamination then those will cluster first. Check to see if that is the case for this particular library. Only remedy is to do a bead clean-up before re-quantification followed by a re-run.

If your provider provides a guarantee for a reasonably uniform distribution of read numbers per sample then they may do a re-run at no cost to you.

ADD REPLYlink modified 18 days ago • written 18 days ago by genomax71k
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