Poor data but why?

[JPC]

Hello, we're new to MiSeq and finding it difficult to relate the SAV information to our protocol to see what's going wrong.

Has anyone seen this sort of pattern (poor but increasing intensity)? We're seeing it regullarly when we use the Nextera XT protocol on long range PCR products but we're seeing much better data with our haloplex work and our home made capture kit so I'm at a loss to explain it.

[GenoMax]

Are you using the newest version of MCS available (http://support.illumina.com/sequenci...wnloads.ilmn)? There is a new version of RTA included in this MCS that addresses problems with low nucleotide diversity data on MiSeq.

[JPC]

We are now, this run was the last one doen on the previous version. So would you say that this looks like a low complexity issue? there are 18 or so long range fragments pooled in our samples, it's probably 150 - 200Kb

[GenoMax]

We are now, this run was the last one doen on the previous version. So would you say that this looks like a low complexity issue? there are 18 or so long range fragments pooled in our samples, it's probably 150 - 200Kb

The quality dropoff in Q-scores heatmap does look similar to what one would see with low nucleotide diversity samples. These issues should be addressed by the new MCS.

[IDDan]

In the DATA by Cycle window change the y axis value to %base...that should give you an idea of diversity. Additionally, your density is a bit high and variable, as is your PF range. We have seen Qscore dropoff for a variety of reasons.

If the issue is diversity, then 2.2 should solve your problem. If your issue is not diversity/complexity then 2.2 probably won't solve it.

[JPC]

Thanks all for your comments. Illumina seem confident that we have an over clustering problem and not a diversity issu, we're going to proceed on that basis and set up another run later this week. I'll post the results

bw
JPC