Two lane failures and low cluster density - expired reagents?

[Turnerac0987]

Hello everyone!

I am currently in the middle of running a 72bp paired end, multiplexed run on a GAIIx. So far, the run is stopped after the first index read, so it's halfway through.

After looking at the run report, I noticed that two of the lanes (3 & 6) didn't have any clusters at all and the remaining lanes, including Phix, had lower than normal data. There was also something weird going on with lane 8. The left side of the pictures had a verticle sharp line and bright clusters while the right side had more dim clusters. I thought it might be a line of oil, but I checked and everything seemed fine.

When I ran the cluster station, all the lanes were flowing fine and nothing seemed out of the ordinary. I called Illumina and they looked at the data. Even they didn't seem to know what was wrong, saying that the intensities looked fine, and overall quality is not horrible.

What bothers me though, is that they seemed to not take the situation as seriously when we found out the cluster kit reagents I was using were expired by 3 months. I was using kits bought by someone else, and the expiration date was removed, so I didn't know until Illumina checked the lot numbers.

So my questions are:
- Can these types of problems really be caused by expired reagents? And do these reagents really expire exactly when Illumina says they do?
- Is there any other explanation that could be causing no clusters in some lanes, and lower data in the other lanes?

I hope I provided enough information. I have a year experience running the GAIIx, but this is my first time troubleshooting, so I don't know all the terminology yet...

Thanks for all your help!

~Angela

[phoss]

Hi Angela,
We had a similar problem to what you outlined (we also ran a multiplexed run on the IIx, several actually). Was the sample concentration in lanes 3 and 6 the same across all other lanes, or did 3 and 6 have differing concentrations?
It is possible to have low/zero clusters or reads in one lane and plenty of reads in another. What we have learned through such troubleshooting is that over-clustering is a problem, hence a possible motive for low/no reads.
Have you changed your multiplex kit since your last run or tried a different one?

[Turnerac0987]

The concentrations in lanes 3 and 6 were the same as all the other lanes.

At first we (and Illumnia) thought that this was an overclustering problem. However, on closer examination, we realized that the SAV images are autocontrasted so we were actually looking at the oligo lawn surface rather than an overclustered flowcell. So it was confirmed that lanes 3 and 6 had no sample present.

I'm still not sure what caused lanes 3 and 6 to fail and all the rest of the data to be about a third of what it should be. I'm continuing with the run but I'm hoping to get some insight on what the problem could be so this doesn't happen in the future.

Does anyone have any experience with using reagents that are expired? Illumina had an expiration date for those reagents at 6 months, but I figured that it wouldn't be an exact date. And now they are saying the reagents expire in a year, not 6 months, so I'm hesitent to believe this was the actual cause...