Sequencing result have a lot of "N"

[xgm-1999]

Hi,all,
I have already sequencing a miRNA library on GA I system, but the reads have a lot of "N", Who can tell me why? thanks!

[floor121]

Only the miRNA library have lot of 'N" in this run? How about the cluster density?

[xgm-1999]

Yes,~21K Clusters/tile

[floor121]

Yes,~21K Clusters/tile

Maybe you could reduce the cluster density, 21k seems a little high...

[xgm-1999]

OK, I will try.

[simonandrews]

Maybe you could reduce the cluster density, 21k seems a little high...

21k is really low on an illumina flowcell and would equate to a total cluster number of only around 2.5 million. 250k raw clusters/tile would be at the upper end of where we'd normally aim to have our cluster density - we'd usually be happier a little below that, but we have had lanes with that kind of density and good results.

If your cluster density was really that low, I'd also be interested to see what your 1st cycle intensity was, and what drop off you got through the run. This may be the root of the basecalling problems you saw.

[basickler]

I'm going to agree with Simon here. Your run probably had cluster station/run issues if the density was that low and I'd guess this is the source of the problems.
Brad