Illumina/Solexa Genome Analyzer Primer/Adapter Sequences

[ECO]

The following sequences were obtained from here (**):

Sequences for Solexa Library Preparations:

Genomic DNA oligonucleotide sequences

Adapters 1
5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT

PCR Primers 1
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

Genomic DNA Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT


DpnII gene expression oligonucleotide sequences

Gex Adapter 1
5' P-GATCGTCGGACTGTAGAACTCTGAAC
5’ ACAGGTTCAGAGTTCTACAGTCCGAC

Gex Adapter 2
5' CAAGCAGAAGACGGCATACGANN
5' P-TCGTATGCCGTCTTCTGCTTG

Gex PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Gex PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Gex Sequencing Primer
5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC

NlaIII gene expression oligonucleotide sequences

Gex Adapter 1
5' P-TCGGACTGTAGAACTCTGAAC
5' ACAGGTTCAGAGTTCTACAGTCCGACATG

Gex Adapter 2
5' CAAGCAGAAGACGGCATACGANN
5' P-TCGTATGCCGTCTTCTGCTTG

Gex PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Gex PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Gex Sequencing Primer
5' CCGACAGGTTCAGAGTTCTACAGTCCGACATG


Small RNA oligonucleotide sequences

RT Primer
5' CAAGCAGAAGACGGCATACGA

5' RNA Adapter
5' GUUCAGAGUUCUACAGUCCGACGAUC

3' RNA Adapter
5' P-UCGUAUGCCGUCUUCUGCUUGUidT

Small RNA PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Small RNA PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Small RNA Sequencing Primer
5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC

**disclaimer blah blah blah. We take no responsibility if you blow a flow cell using these sequences!

[sci_guy]

Bravo! Just what I needed.

[horigen]

Very useful!
I refined these sequences. Hopes they're clearer.


Genomic DNA

Adapter:
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3'
3' -------------------- -----TGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGp (-) -------------------- -------------- 5'
Adapter:
5' -------------------- -------------------- ------------------ (-) pGATCGGAAGAGCTCGTATG CCGTCTTCTGCTTG 3'
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'
PCR Primer:
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3'
3' -------------------- -------------------- ------------------ (-) -------------------- -------------- 5'
PCR Primer:
5' -------------------- -------------------- ------------------ (-) -------------------- -------------- 3'
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'
Result Library:
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (N) AGATCGGAAGAGCTCGTATG CCGTCTTCTGCTTG 3'
3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGA (N) TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'
Genomic DNA Sequencing Primer:
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3'
3' -------------------- -------------------- ------------------ (-) -------------------- -------------- 5'

DpnII gene expression

Gex Adapter 1:
5' -------------------A CAGGTTCAGAGTTCTACAGT CCGAC--------------- -------------------- ------ 3'
3' -------------------- ---CAAGTCTCAAGATGTCA GGCTGCTAGp---------- -------------------- ------ 5'
Gex Adapter 2:
5' -------------------- -------------------- -------------------- ----pTCGTATGCCGTCTTC TGCTTG 3'
3' -------------------- -------------------- -------------------- ---NNAGCATACGGCAGAAG ACGAAC 5'
Gex PCR Primer 1:
5' -------------------- -------------------- ----------------------------------------- ------ 3'
3' -------------------- -------------------- -------------------- -----AGCATACGGCAGAAG ACGAAC 5'
Gex PCR Primer 2:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGA---------------- -------------------- ------ 3'
3' -------------------- -------------------- -------------------- -------------------- ------ 5'
Result Library:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGACGATCNNNNNNNNNNN NNNNNTCGTATGCCGTCTTC TGCTTG 3'
3' TTACTATGCCGCTGGTGGCT GTCCAAGTCTCAAGATGTCA GGCTGCTAGNNNNNNNNNNN NNNNNAGCATACGGCAGAAG ACGAAC 5'
Gex Sequencing Primer:
5' -----------------CGA CAGGTTCAGAGTTCTACAGT CCGACGATC----------- -------------------- ------ 3'
3'--------------------- -------------------- -------------------- -------------------- ------ 5'

NlaIII gene expression

Gex Adapter 1:
5' -------------------A CAGGTTCAGAGTTCTACAGT CCGACATG------------ -------------------- ------ 3'
3' -------------------- ---CAAGTCTCAAGATGTCA GGCTp--------------- -------------------- ------ 5'
Gex Adapter 2:
5' -------------------- -------------------- -------------------- ----pTCGTATGCCGTCTTC TGCTTG 3'
3' -------------------- -------------------- -------------------- ---NNAGCATACGGCAGAAG ACGAAC 5'
Gex PCR Primer 1:
5' -------------------- -------------------- -------------------- -------------------- ------ 3'
3' -------------------- -------------------- -------------------- -----AGCATACGGCAGAAG ACGAAC 5'
Gex PCR Primer 2:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGA---------------- -------------------- ------ 3'
3' -------------------- -------------------- -------------------- -------------------- ------ 5'
Result Library:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGACATGNNNNNNNNNNNN NNNNNTCGTATGCCGTCTTC TGCTTG 3'
3' TTACTATGCCGCTGGTGGCT GTCCAAGTCTCAAGATGTCA GGCTGTACNNNNNNNNNNNN NNNNNAGCATACGGCAGAAG ACGAAC 5'
Gex Sequencing Primer:
5' ----------------CCGA CAGGTTCAGAGTTCTACAGT CCGACATG------------ -------------------- ------ 3'
3' -------------------- -------------------- -------------------- -------------------- ------ 5'

Small RNA

5' RNA Adapter:
5' -------------------- ---GUUCAGAGUUCUACAGU CCGACGAUC (-) -------------------- - 3'
3' -------------------- -------------------- --------- (-) -------------------- - 5'
3' RNA Adapter:
5' -------------------- -------------------- --------- (-)pUCGUAUGCCGUCUUCUGCUU GUidT 3'
3' -------------------- -------------------- --------- (-) -------------------- - 5'
RT Primer:
5' -------------------- -------------------- --------- (-) -------------------- - 3'
3' -------------------- -------------------- --------- (-) AGCATACGGCAGAAGACGAA C 5'
Small RNA PCR Primer 1:
5' -------------------- -------------------- --------- (-) -------------------- - 3'
3' -------------------- -------------------- --------- (-) AGCATACGGCAGAAGACGAA C 5'
Small RNA PCR Primer 2:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGA----- (-) -------------------- - 3'
3' -------------------- -------------------- --------- (-) -------------------- - 5'
Result Library:
5' AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGT CCGACGATC (N) TCGTATGCCGTCTTCTGCTT G 3'
3' TTACTATGCCGCTGGTGGCT GTCCAAGTCTCAAGATGTCA GGCTGCTAG (N) AGCATACGGCAGAAGACGAA C 5'
Small RNA Sequencing Primer:
5' -----------------CGA CAGGTTCAGAGTTCTACAGT CCGACGATC (-) -------------------- - 3'
3' -------------------- -------------------- --------- (-) -------------------- - 5'

Two errors have been corrected. (Genomic DNA Adapter & Small RNA Result Library)
I'm sorry for that and other potential errors.

[ECO]

Thanks Horigen! That definitely clarifies things!

If anyone is having trouble viewing, make sure your browser is as wide as it can go.

[tec]

Hello,
just one question.

We want to synthesize the adapters by ourselves.
Its necessary to phosphorylate the 5' end of the adapters additional?
(there is still no 5' phosphate?)

Thanks a lot!

[sci_guy]

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

[dandestroy]

Sorry for the small font, but that's the only way I could make it fit


Paired-end DNA

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'

[sigusn]

Can someone explain to me why the Illumina adapters need to be 5´-phosphorylated? In the very similar SOLiD sample prep protocol the adapters are not phosphorylated.

[sci_guy]

Illumina adaptors need 5' phosphorylation for the ligation. The newer SOLiD protocol uses a "nick translation" like step where the "nick" left over from ligating primers without a 5'-phosphate is translated to (or near enough to) the primer terminus. One strand will ligate as there is a 5' phosphate on the end repaired template DNA. The SOLiD adaptors are blunt-ended on the termini designed for ligation, whereas the Illumina adaptors use a T overhang to create incompatible ends. If the SOLiD primers had 5' phosphate groups there would be a LOT of P1-P2, P1-P1 or P2-P2 ligated product; hence the nick translation like step. Nice clever twist.

[swaroom]

What conditions do I use to anneal the adapters and what concentration is optimal for ligation of adapters to the fragmented genomic DNA. I am ordering the Illumina primers through Invitrogen. :

[Jenny Russ]

mRNA-Seq adapter sequences?

The sequences above are really helpful. Does anyone also have the sequences for mRNA-Seq? This would be really great!!!

[Joanne Ho]

I use PE-adapter to mRNA-seq, it can work properly.

[monad]

mRNA-seq adapters

The sequences above are really helpful. Does anyone also have the sequences for mRNA-Seq? This would be really great!!!

mRNA-seq kit uses PE adaptors, and read 1 primer is same as genomic DNA primer for single read sequencing. If you have a PE sample ready, but whatever the reason wants single end sequencing, do that just like for SR sequencing. Done that many times.

[berthasalco]

Hello there,

first at all, i want to thank you to all to coperate to give us the sequence of the GEX 1 and 2 adapters and primers for the tag profilling with DpnII , but

does anybody know at which concentration are they used??? The Illumina protocol does not give concentrations. According to the Invitrogen LongSAGE protocol the double stranded adapters have a concentration of 40 ng/ul and 1.5 ul are used for one ligation reaction

thanks a lot,
BS

[bioinfosm]

Hi all,

Thanks for the technical details on this. Anyone got some information about bioinformatics on adapter contamination detection and removal. I tried using adapter sequences with eland to check for contamination, but less than 1% reads aligned. I expected more as less than 10% reads aligned to actual reference.

I know of a file one can specify in GA pipeline, to exclude sequences -- could someone point more details on the same? (i tried bioinformatics thread, but did not hear back )

thanks!