5' end of Illumina sequencing primers, specificity

[MaxNorkin]

Hello to everybody!

I am new in Illumina sequencing and have a question (apology if the answer is already somewhere here, but I could not find it)

I had a look on Illumina sequencing primers and the primers which used for library preparation (Nextera) (https://support.illumina.com/content...0002694-01.pdf, http://nextgen.mgh.harvard.edu/attachments/Nextera%20Protocol.pdf) and it seems that they have just 3' end complementarity (19 out of 38 nt). In theory 3' end part would be enough for specific Sequencing by Synthesis. Why do you need this non-complementary 5' tail then? Why don't make it shorter? Or am I just looking at some wrong sequences?

Here are the sequencing primers for the library prepared with Nextera Kit:
Nextera Read 1 Primer: 5′-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3′
Nextera Read 2 Primer: 5′-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3′

Final product ends:
Fw: 5’-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[GSP part]
Rev: 5’- CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[GSP part]

Attached there is a nicer picture

Thanks for help!

[ianakiev]

Originally Posted by MaxNorkin

Hello to everybody!

I am new in Illumina sequencing and have a question (apology if the answer is already somewhere here, but I could not find it)

I had a look on Illumina sequencing primers and the primers which used for library preparation (Nextera) (https://support.illumina.com/content...0002694-01.pdf, http://nextgen.mgh.harvard.edu/attachments/Nextera%20Protocol.pdf) and it seems that they have just 3' end complementarity (19 out of 38 nt). In theory 3' end part would be enough for specific Sequencing by Synthesis. Why do you need this non-complementary 5' tail then? Why don't make it shorter? Or am I just looking at some wrong sequences?

Here are the sequencing primers for the library prepared with Nextera Kit:
Nextera Read 1 Primer: 5′-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3′
Nextera Read 2 Primer: 5′-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3′

Final product ends:
Fw: 5’-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[GSP part]
Rev: 5’- CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[GSP part]

Attached there is a nicer picture

Thanks for help!

You need these "Long" primers in order to be able to cluster/sequence. Cheers

[MaxNorkin]

Dear ianakiev, thanks for your reply! I am sorry, but I think you didn't get my question. I am asking about sequencing primers, not indexing primers. These ones:

Nextera Read 1 Primer: 5′-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3′
Nextera Read 2 Primer: 5′-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3′

I didn't get why there are some strange non-complementary (to cDNA library sequences) letters at the 5'ends of these primers.


Anyway, it seems that these are wrong sequences, real sequencing primers should have the same sequence as transposes adapters (the sequence between GSP and Index). Well, it's an assumption because Illumina doesn't provide this information (according to our CF). I am wondering if somebody performed Sanger sequencing of these guys?

[JoeKutch]

I think what he's trying to say is that those are the sequences needed to actually bind the template to the oligonucleotide lawn on the flow cell.

[MaxNorkin]

Oh.. I am sorry if I misunderstood. Now I'm stuck. Could you please give a hint how that would work? How sequencing primers could help to bind the template to flow cell?

I thought that cDNA molecules bind to the flow cell via P5/P7 adaptors which are located at the very end of template. These ones:

Final product ends:
Fw: 5’-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[GSP part]
Rev: 5’- CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[GSP part]

(?_?)

[JoeKutch]

Sorry - it looks like I misunderstood, not you.

I think the added sequencing primer bases are meant to keep the Tm of the primer in the appropriate range for the sequencer. For example, when designing custom primers, you need to aim for a Tm above 60-65 (it varies a little by sequencer) to ensure that it anneals and doesn't melt away during synthesis.

[ianakiev]

The sequences in green colors are needed for hybridization to the flow cell primers. That is what I meant. Cheers

[saurabhiitd]

The reason they have such sequence this that during ligation or transposon based insertion. They will form a Y shaped fork.
>= XXXXXXXXXXXXXXXX =< where XXXXXXXXXXX is your insert
= represents the complementary sequence between Adaptor 1 and Adaptor 2 (highlighted in orange in your question). > and < represent two strands of Adaptor 1 and 2 which do not have complementarity.

Adaptor 1 may have the exact sequence for say Primer 1 whereas Adaptor 2 will have the reverse complementary sequence to Primer 2.
or
Adaptor 2 may have the exact sequence for say Primer 1 whereas Adaptor 2 will have the reverse complementary sequence to Primer 1.
Please look at the adaptor sequences and you would realize this.
I am not 100% about Nextera primers but this is a generally Illumina strategy for many sequencing kits.