[nanos]

hi!!

i would like to use custom adaptors to do ChiP sequencing.

for this i would need to know what the concentration of the adaptor for this protocol is.

thanks for your help

[AdamB]

Quick FYI. I spent many hours trying to get the PCR amplification working with Sigma primers (with the phosphorothioate bond), but with very low yields. After switching to the Illumina-supplied primers, my yield shot up.

What suppliers for primers have people used, where they found the custom primers to work properly?

[kmcarr]

What suppliers for primers have people used, where they found the custom primers to work properly?

IDT
(now some jibberish to meet the 10 character minimum for posting)

[NextGenSeq]

I don't see anyone mentioning the two locked nucleic acid (LNA) bases in the primers. People have told me that this dramatically increases library yields.

http://genomics.med.tufts.edu/documents/htseq_protocol_for_illumina_paired.pdf

[AdamB]

IDT
(now some jibberish to meet the 10 character minimum for posting)

Thanks, and did you have the phosphorothioate bonds at the 3' end?

[clz8782]

The P.E adaptors/primers have been added at the original link.

I have ordered Illumina's paired end primers and had low yield of PCR amplicons of my library. I was sent supplemental information from this nature paper. http://www.nature.com/nature/journal...re07517-s1.pdf

This states that the PCR primers should have a phosphorothioate bond at the 3' end:

5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGC
TCTTCCGATCxT and 5’-
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT
CCGATCxT (x = phosphorothioate bond resistant to excision by 3’-5’ exonucleases).

This is the first I am hearing of this. Should the Paired End PCR Primers be modified in such a way?

[pedstunite]

overhang on adapters not for ligation purposes

Hi,

I have the same question regarding the adapter sequence. I can't understand why there is an overhang on the 5' end of an illumina adapter sequence (not the overhang needed for ligation). B/c this sequence is amplified by PCR anyways, I don't see the need for an overhang. Anyone know???

Thx

[NextGenSeq]

I have ordered Illumina's paired end primers and had low yield of PCR amplicons of my library. I was sent supplemental information from this nature paper. http://www.nature.com/nature/journal...re07517-s1.pdf

This states that the PCR primers should have a phosphorothioate bond at the 3' end:

5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGC
TCTTCCGATCxT and 5’-
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT
CCGATCxT (x = phosphorothioate bond resistant to excision by 3’-5’ exonucleases).

This is the first I am hearing of this. Should the Paired End PCR Primers be modified in such a way?

I believe this is because they use proof-reading polymerase in the PCR reaction. We use locked nucleic acids instead of phosphorothioate modifications since this is what the Tufts protocol recommends. If you don't modify the primers the yield will be much lower.

[edge]

Hi,

Does anybody got the idea regarding the adaptor sequence use for small RNA seq data of ILLUMINA IDEA CHALLENGE?

http://www.illumina.com/landing/idea/

Thanks a lot for sharing.

[edge]

From the following link, http://intron.ccam.uchc.edu/groups/t...Sequences.html

Small RNA oligonucleotide sequences
5' RNA Adapter
5' GUUCAGAGUUCUACAGUCCGACGAUC

3' RNA Adapter
5' P-UCGUAUGCCGUCUUCUGCUUGUidT

Do I need to convert the "U" to "T" before I start trimming the adaptor sequence in my raw data?
Besides that, what is the "id" in 3' RNA Adapter refer to?
Thanks for any advice.

[MW007]

tru-seq adapters?

Does anyone know if the new tru-seq system uses different adapters; i.e. different sequences than those posted below?

[genlyai]

MW007, I've heard truseq does use new seqs for some oligos, but I'm not sure which. If anyone can answer this -- possibly we can get a start just from looking at a run that's used Truseq -- that would be great.

Another thing I didn't see on here is sequences of the barcode primers (from pre-truseq days). Can anybody help with that?

[seqgirl123]

anyone use qPCR primers during library prep?

Does anyone use the qPCR primers sold by Applied Biosystems to amplify their PCR product? Their qPCR primer sequences they sell are identical to Illumina's flowcell oligos 'P5' and 'P7'.

Taking an example, say you have a genomic paired-end library and for whatever reason, there's low library yield after PCR cleanup. So you decide to reamplify. But instead of reamplifying with the primers again (which is the common route), does anyone use the qPCR primers instead? The reasoning behind it is that your library now contains the regions complementary to flowcell oligos, so the qPCR primers can be used for reamplification instead of the PE-primers. I am not sure how this would help though. Perhaps because the qPCR primers are same as flowcell oligos, they will amplify the material better and thus will lessen PCR bias, and give higher quality sequencing data? Has anyone ever heard of this?

On another note, I haven't heard of people using the qPCR primers in place of PE primers however, and not sure if it can be done either. I haven't checked it out myself.

[NextGenSeq]

We use the KAPA library quantification kit. It comes with everything you need and works very well.

[seqgirl123]

Do you ever use the KAPA GAIIx qPCR primers for library re amplification before putting the library onto the cluster station?