454 Library prep for limited sample

[TonyBrooks]

I have a few samples with less than 500ng (some as low as 200ng) of DNA that we would really like to prep for Titanium sequencing.
Has anyone attempted this before, and if so, did you generate decent libraries that produced decent sequencing?

Did you modify the protocol in any way?

Things I'm thinking of;
a) Covaris shearing as opposed to nebulisation
b) Gel-based size selection (e-gel) instead of beads
c) PCR amplification of final library

Appreciate any pointers...

[MrGuy]

Read these:

http://www.ncbi.nlm.nih.gov/pubmed/21886102

http://www.ncbi.nlm.nih.gov/pubmed/20435675

[Zaag]

I made good libraries with 100 ng starting DNA.

1) Yes, use the Covaris.
2) No, use beads.
3) Only necessary if the final concentration is too low to measure or run on a bio-analyzer.

[TonyBrooks]

I made good libraries with 100 ng starting DNA.

1) Yes, use the Covaris.
2) No, use beads.
3) Only necessary if the final concentration is too low to measure or run on a bio-analyzer.

Hi Zaag
Can you let me know your Covaris settings? Our libraries always seem to be on the large size (800-1000bp). We've tried the suggested settings for both 500bp and 400bp shearing.

[Zaag]

we use the covaris S2:

45 seconds
Intensity 3
Dutycycle 5%
Cycleburst 200

use the tubes from covaris, not eppendorfs or such
fill up your sample with TE or EB of H2O or whatever till the tube is full (130 ul)

we routinely have gDNA from blood isolated by a gentra and it works fine for that, however we do see that if DNA is isolated manually the time can differ; settings remain the same;

hope this helps

[Zaag]

by the way, as for point 3, maybe performing a few cycles of PCR is not a bad thing because you measure fragments that you can not use for emPCR: the libraries with only 1 adapter

[TonyBrooks]

I used the same settings but 90 second shearing and 120µL on our S2 with the AFA snap cap tubes. Increased time should generate smaller fragments, surely?
I wonder why we're getting large libraries.

We're going to quantify our libraries by qPCR, so no need for enrichment PCR. I never liked the Roche 454 fluorescence assay because, as you say, it doesn't account for single adapter ligation.