[MW007]

tru-seq adapters?

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

[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.

[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.

[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.

[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

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?

[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?

[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

[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)

[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?

[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

[[email protected]]

PE indexed libraries

I'm trying to figure out what the final sequence of an Illumina PE indexed library is and where the enrichment primers and sequencing primers go. Has anyone figured out a schematic of generation of the PE indexed libraries showing all the sequence similar to the one I found on this forum for non-indexed PE libraries (see below).

dandestroy09-18-2008, 07:13 PM
Sorry for the small font, but that's the only way I could make it fit




PE Adapter1:
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
3' -------------------- -----TGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGp (-) -------------------- -------------------- -------------------- - 5'
PE Adapter2:
5' -------------------- -------------------- ------------------ (-) pGATCGGAAGAGCGGTTCAG CAGGAATGCCGAG------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTC------- -------------------- - 5'
PE PCR Primer1:
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5'
PE PCR Primer2:
5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5'
Result Library:
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (N) AGATCGGAAGAGCGGTTCAG CAGGAATGCCGAGACCGATC TCGTATGCCGTCTTCTGCTT G 3'
3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGA (N) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5'
PE DNA Sequencing Primer1
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5'
PE DNA Sequencing Primer2
5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGC--- -------------------- - 5'

[AdamB]

I ordered the following primers from Sigma:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T

CAAGCAGAAGACGGCATACGAGCTCTTCCGATC*T

(HPLC-purified)
*=Phosphorothioate bond

However I can't get them working with my ChIP input DNA sample. Have tried varying the number of cycles, but each time after the PCR amplification of my library I only have 2 or 3 ng/ul, whereas colleagues have had more like 20 ng/ul.

I'm unsure whether the problem is with the primers or something else. As far as I know, everything apart from the primers is the same as colleagues' libraries that worked. I don't have any Illumina primer to test alongside.

[mx1970]

Hi,
I want to do directional mRNA seq. I have to use the Small RNA Sequencing Primer during the primer hybridization.
Does anybody know at which concentration should I adjust this primer?
thanks

[amango]

I noticed that several people were wondering about adaptor and primer concentrations in the Illumina kit. I just spoke to an Illumina tech support person, and for RNA-seq at least, the working concentration for the adaptor oligo mix is 15 uM while PCR primers are 25 uM.