Deep Seq Barcode 'crossover'

[JPC]

Hello, we have just completed a run with 17 barcodes in a single E120. Three samples have deep coverage for one set of genomic fragments, 14 have deep sequence for a different set of fragments. Where depth gets up to and over 5,000x we start to see that fragment pop up in all other samples, we're looking at a 0.002 - 0.003% cross over.

Does anyone see anything similar in their high coverage samples?..perhaps you haven't noticed it?

Lab contamination was our first thought (and we're not ruling that out yet); however, as there were 17 samples, one of the barcodes (#17) came from an unopened kit, we had never used #17 before. Also the two sets of fragment libraries were created weeks apart so I could accept the first set contaminating the second I can't see how both could contaminate each other.

If this were random mis-calling of barcodes or random sequnce error then we would see the 'cross over' to barcodes that were not used ......and we don't.

Could there be any sort of mix up going on in the emPCR? carried over barcodes?..... What about in software? could there be a binning problem with high coverage fragments?

JPC

[JPC]

A correction from my last post. The cross over is apparent in barcodes beyond the 17 that we have used, so perhaps we are seeing mis-calling or a barcode binning problem?

[snetmcom]

what level of mismatch tolerance do you have set for barcode binning?

[JPC]

Hi snetmcom, thanks for your input.

The value is set to 0, this is not something we have ever changed and in fact this if the first I have heard of it!

JPC

[colindaven]

I wouldn't change that mismatch tolerance value in a hurry. 0 sounds like a good idea.

Thanks for the warning JPC.