Fragment length limits?

There must be a size limitation due to how much DNA can be put onto their ionspheres, i.e. longer templates have less on the ionsphere, produces less H+

this would be my guess as well. High copies per sphere until their algorithms get better?

[The_Dark_Base]

The small insert size really voids the use of the PGM for their often touted library validation application. Looking at the tech note, you have a library with adapters, then add an oligo on to attach to their particle. With all that only adapter dimers would work on the PGM?

There must be a size limitation due to how much DNA can be put onto their ionspheres, i.e. longer templates have less on the ionsphere, produces less H+

Just look at binding on other Life Technology spheres:

"The amount of biotinylated DNA immobilized will depend on fragment size. Due to steric hindrance, the binding efficiency is significantly reduced when the fragment size exceeds..."

http://tools.invitrogen.com/content/sfs/gallery/high/3612.jpg

[Marzanna]

We are still waiting for installation so I can only share theoretical knowledge from the secret documents ;-)

The fragmentation is done with BioRuptor or Covaris (both described).
"If the size distribution of the fragmented DNA is in the 150–200 bp range, you can use alternative fragmentation methods such as the Covaris System."

After end repair and adapter ligation there is size selection step (eg. Pippin Prep).
"For optimal sequencing results, size-select a DNA library with a mean size of 185–210 bp, with a size distribution of ±20 bp around the mean. Libraries with a mean size >~220 bp yield results of reduced sequencing quality."

I was also told that too wide insert size range (like size selection on beads for SOLiD) leads to too high percentage of short reads (50-100bp).

And now I will be taken directly to the IonHell
see you around
Marzanna

[krobison]

I've been told that with the Ion Fragment Kit, the maximum input fragment size should be 150 bp so that with adapters the fragment is no longer than 210 bp.

Can anyone with access to Ion's deepest, darkest secret documents (known elsewhere as "package inserts") confirm this? The application note on Amplicon Sequencing mentions nothing on the subject.

Anyone know if the constraint is imposed in the library prep or the emPCR template prep? What imposes it? Extension times? Nucleotide concentrations? emPCR micelle sizes?

One might guess this is something Ion will need to address in order to hit 454-like read lengths