shorter read lengths than anticipated

[chi_bet740]

Hi All,

I'm currently using the Lib-L kit for GS Junior. My PCR product size is around 500 bp, but I've been having trouble getting sequence reads with that length...After the sequencing run, I saw several read length distributions, for example, a peak at 150 bp, 300 bp, 400 bp, and 500 bp. Previously, I've also seen other samples that had two peaks, around 400 and 500 bp. These were all done with shot gun processing - so when I convert the raw data from shot gun to amplicon sequencing, I could still get enough sequences for analysis, but I'd like to resolve the issue since our lab has been using shot gun processing. Is there any step in the library prep I should be aware of to improve my read lengths? Size check with flashgel after ligation step indicated that I did have the correct fragment size.

Thanks for your help!

[Torst]

Maybe this is a silly question, but if your DNA sample is a PCR product of ~500bp, can't you skip the 'shotgun' process? No shearing is required as it is already at the target size?

[Aniki]

Hi All,

I'm currently using the Lib-L kit for GS Junior. My PCR product size is around 500 bp, but I've been having trouble getting sequence reads with that length...After the sequencing run, I saw several read length distributions, for example, a peak at 150 bp, 300 bp, 400 bp, and 500 bp. Previously, I've also seen other samples that had two peaks, around 400 and 500 bp. These were all done with shot gun processing - so when I convert the raw data from shot gun to amplicon sequencing, I could still get enough sequences for analysis, but I'd like to resolve the issue since our lab has been using shot gun processing. Is there any step in the library prep I should be aware of to improve my read lengths? Size check with flashgel after ligation step indicated that I did have the correct fragment size.

Thanks for your help!

Hello, in the last several 454 runs on our FLX I have noticed this as well. Our FAS is also unsure of why this is happening. I suggest you ignore the issue and just do as you have always done. This is "normal" currently. There are some issues out there with the 454 kits, this can be one of the outcomes.

I am sorry I was not able to help you but this is an ongoing issue.