problems with 454 library prep

[obsidiana]

I’m preparing the library for genomic DNA following the 454 protocol. However by Agilent analysis for library I’m seeing some strange 1 to 2kb long fragments beside expected 800-1000pb peak.
Has anyone encountered a similar problem?

[LMcSeq]

There's been rumor that the adapter in the rapid library kit makes the libraries look much longer because of the FAM label. Try running the adapter alone on the Bioanalyzer and see what it looks like. We've seen this on many occasions and sequenced the longer libraries with good results. I'm not sure about that shouldering that you have on the right side of the library though....haven't seen that before, but I bet you can get good sequence out of it.

[pmiguel]

I’m preparing the library for genomic DNA following the 454 protocol. However by Agilent analysis for library I’m seeing some strange 1 to 2kb long fragments beside expected 800-1000pb peak.
Has anyone encountered a similar problem?

Which protocol? Standard or Rapid? If Standard, are these a shotgun or paired end library.

--
Phillip

[Soulbee]

I have also prepared cDNA libraries using rapid library kit and protocol with MID adaptors. I actually elongated the chemical fragmentation step to 1 minute instead of 30 seconds because of the large (avg ~1300bp) fragment sizes. Still, the final library's average fragment size was around 1000bp. any thoughts?

[LMcSeq]

We've seen longer libraries in general with the rapid kit. I don't think it would be any different between the regular and cDNA kits.

[RCJK]

I've seen the same thing with many of my Rapid Libaries. They've sequenced fine though. What I've also noticed about the HS DNA Bioanalyzer chips is a lot of inconsistency. I can run the same library prep multiple times (on the same chip and across multiple chips) and I'll get a different looking result each time. I haven't had a chance to follow up w/ Agilent about this yet though. Also, it seems as though when I run a sample that has a lot of large fragments, similar to the one in the picture above, it affects the following one or two samples and they tend to not run properly. If those following samples are then rerun on a new chip or after a couple of blank wells, they will look fine.

[obsidiana]

Phillip, this is the rapid protocol, a shotgun library.

I’ve also notice the Bioanalyser chip inconsistency but in my opinion this is not the case, this seems to be more a fragmentation problem.

We have prepared new libraries with an elongation in the fragmentation step, 1 minute 15 seconds instead 1 minute. The strange big fragments have disappeared and the average fragment size was fine.

We will try to sequence!