recent decrease in 454 FLX + read length

[lhandley]

We upgraded our 454 to FLX+ in August 2011 and have been running several runs per week with much longer average read lengths (>600bp) than our Titanium FLX runs (350bp). However, at the beginning of November the FLX+ kits were put on backorder due to "quality issues" - the kits were not passing specs set by 454. At the beginning of December, we started to receive kits again and have run 8 wgs runs since. All of these runs have a much shorter average read length (~420bp) than what we were getting previously. Has anyone else received FLX+ kits recently that yielded much shorter reads?

[MissDNA]

We haven´t had our upgrade yet but more I read about it, more worried I get. Do you still perform regular Titanium runs? Are they all right?

Is only the library that shows shorter avg reads, or the controls are shorter too?

For several months we had problems with short reads, at first we thought it was a bad lot of XLR70, turned out or equipment had some problem, one part was replaced and things went back to normal.

[SeqAA]

Have you tried running some of your previous samples to rule that out?

[lhandley]

The new runs were old libraries that I had already sequenced several times - just trying to get more sequence data. Plus, I ran 2 new libraries to make sure that it wasn't an issue with trying to run "old" libraries. Just seems that everything is shorter than it was before with the first lots of reagents we received.

[RCJK]

lhandley, are the kits that were used for the runs that are giving you shorter reads a new lot of XL+ kits? I have had a couple of great XL+ runs, but fear that after the quality issues and backorder that a new batch of kits may not perform as well.
Has Roche given you any feedback about this? Running a library that previously yielded good results would definitely be worth considering if it's an option.

[lhandley]

Yes, these are a new lot of FLX+ kits that we received after the backorder. I am running the same libraries as before, so that is why I have noticed the drop in read length so easily - before I was getting close to 600bp avg. read length for these libraries, now it is much lower. Roche is looking into all of my sequencing data now, I was just wondering if anyone else had noticed this drop in read length using kits that they have received after the backorder situation.

[pmiguel]

lhandley,
Have you contacted GS-Support? Sometimes it can be hard to get through and they seem to be massively over-tasked. But they have access to tools to troubleshoot your run. It could be a batch issue, but it could also be an instrument issue. (For example, it seems the heating plate control is much more critical to get good FLX+ runs, than it was for FLX.)

--
Phillip