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Old 01-17-2018, 01:08 PM   #15
Location: Storrs, CT

Join Date: Sep 2012
Posts: 13

I think it would be helpful to us to know where you got your indices from, as nucacidhunter mentioned. I don't know if it's possible, but perhaps the indices from Illumina are selected because they work very well on the HiSeq. Since the way that they cluster is different, I *could* see it being possible that certain sequences on the HiSeq perform better than on the MiSeq. But I really can't imagine why that would be so, unless certain parts of the sequence upstream of the flow-cell adapter are better for the recombinase enzyme that the HiSeq uses?

It could also be a function of the libraries themselves, and NOT the indices. Were your libraries all of uniform size distribution? I've observed that the MiSeq can be very sensitive to the presence of long (>800bp) inserts -- those molecules simply won't cluster as well as the shorter inserts. If the poor-on-MiSeq library had larger inserts than the other libraries, that could explain why it didn't form as many clusters on the MiSeq flowcell, but was able to form clusters well on the HiSeq.

I feel your frustration. Clustering is some kind of mystical voodoo to those of us who don't work at Genomics cores and don't have tons of experience with the way different libraries cluster on different platforms. I'm dealing with this myself right now -- wide spread of representation over 50+ libraries, only *partly* explained by an aggressive Pippin prep.
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