Hi,
I would like to prepare an enriched (custom IDT lockdown probes) NGS (Illumina platform) library that has a PCR step as late as possible in the protocol - I want to bisulfite treat the already adapter tagged and enriched library or part of it.
Ideally, I would be able to add adapters, use IDT probes, bisulfite treat 2/3 of the library, amplify with PCR, and then in 24 multiplex run the library on Illumina platform. I want to do this separately for bisulfite treated and non-treated parts of the library.
Input to protocol would be a Covaris sonicated human genome DNA, cut to 800 - 1000bp
For that, I guess, I would need a kit that has the following characteristics (please comment if you think they are unnecessary or perhaps unrealistic):
1. High DNA input (1-2 micro gram of starting DNA or more), since the enriched target is relatively small.
2. Adapters which have the cytosins methylated (to resist bisulfite), but are not biotinylated (to not be pulled by the enrichment streptavidin beads).
3. No PCR before I can bisulfite treat (right after enrichment step)
I see that Illumina TruSeq DNA PCR-Free LT
(http://support.illumina.com/sequenci...ee-lt-kit.html)
satisfies my needs, except I don't know if adapters are biotinylated.
It bothers me that in their webinar of kit comparison
(http://support.illumina.com/content/...ry.html?iframe )
the TruSeq DNA PCR-Free LT is marked as non enrichment-compatible.
QUESTION 1:
Does anybody know, if the adapters in TruSeq DNA PCR-Free LT are biotinylated?
QUESTION 2:
Does anybody know of any other NGS library prep kits that would satisfy my needs even better or for a lesser price?
Enrichment has to be before bisulfite treatment and as far as I know, IDT probes require the presence of adapters, so please don't suggest bisulfite treating before library preparation.
Thanks a million,
Lovro
I would like to prepare an enriched (custom IDT lockdown probes) NGS (Illumina platform) library that has a PCR step as late as possible in the protocol - I want to bisulfite treat the already adapter tagged and enriched library or part of it.
Ideally, I would be able to add adapters, use IDT probes, bisulfite treat 2/3 of the library, amplify with PCR, and then in 24 multiplex run the library on Illumina platform. I want to do this separately for bisulfite treated and non-treated parts of the library.
Input to protocol would be a Covaris sonicated human genome DNA, cut to 800 - 1000bp
For that, I guess, I would need a kit that has the following characteristics (please comment if you think they are unnecessary or perhaps unrealistic):
1. High DNA input (1-2 micro gram of starting DNA or more), since the enriched target is relatively small.
2. Adapters which have the cytosins methylated (to resist bisulfite), but are not biotinylated (to not be pulled by the enrichment streptavidin beads).
3. No PCR before I can bisulfite treat (right after enrichment step)
I see that Illumina TruSeq DNA PCR-Free LT
(http://support.illumina.com/sequenci...ee-lt-kit.html)
satisfies my needs, except I don't know if adapters are biotinylated.
It bothers me that in their webinar of kit comparison
(http://support.illumina.com/content/...ry.html?iframe )
the TruSeq DNA PCR-Free LT is marked as non enrichment-compatible.
QUESTION 1:
Does anybody know, if the adapters in TruSeq DNA PCR-Free LT are biotinylated?
QUESTION 2:
Does anybody know of any other NGS library prep kits that would satisfy my needs even better or for a lesser price?
Enrichment has to be before bisulfite treatment and as far as I know, IDT probes require the presence of adapters, so please don't suggest bisulfite treating before library preparation.
Thanks a million,
Lovro