General Illumina Questions/Primer Design Help

[kayleighepps]

Hi!

I've previously trawled through a variety of information sources and websites trying to understand which sequences/methods Illumina are currently using for single-index multiplex sequencing but I think i've confused myself!

Firstly: In-regards to the paired-end sequences (listed here: http://supportres.illumina.com/docum...enceletter.pdf) - how do these ensure sequencing directionality when the Rd1/2 sequencing primers will technically bind to both sequences due to the partial complimentarity. Is the sequencing reaction done at >60C to ensure high strictness such that full length correct-pairing is required?

I will be soon conducting an experiment requiring deep-sequencing. The product of my experiment will be short ~120bp ssDNA molecules resulting from the ligation of 2 60bp primers (one forward and one reverse). I was wondering if it was at all feasible to design these primers to contain Read1/2 tails - an opposing tail for FOR/REV. This would generate ssDNA molecules with the structure:

5'-READ1-N(120)-READ2-3'

Could I then just amplify these using standard Illumina PE-primers to include an index and the P5/P7 attachment sites?

Are there any special considerations for doing this?

Thank you for any help you can offer!!

Kind regards,

- K

[rajasereddy]

Hi K

Illumina uses same sequence as primer for read 1 and read 2 but the trick is its orientation read 1 primer orientation is exactly opposite to read 2 primer orientation so it won't bind.

When you say sequencing reaction I assume you are mentioning base incorporation if it is so the no the incorporation won't happen >60.

In your case you can use the primers with illumina adapter overhangs to get amplicon product but the problem is it can't be sequenced on illumina machine because of their cluster identification algorithm. You need to have at least random bases for at least first 4 cycles. Better talk to them once.

Cheers
Reddy

[kayleighepps]

Hi K

Illumina uses same sequence as primer for read 1 and read 2 but the trick is its orientation read 1 primer orientation is exactly opposite to read 2 primer orientation so it won't bind.

When you say sequencing reaction I assume you are mentioning base incorporation if it is so the no the incorporation won't happen >60.

In your case you can use the primers with illumina adapter overhangs to get amplicon product but the problem is it can't be sequenced on illumina machine because of their cluster identification algorithm. You need to have at least random bases for at least first 4 cycles. Better talk to them once.

Cheers
Reddy

Thank you for your reply

I do not understand why cluster identification algorithm's will interfere with this. My products will have the following structure:

5'-READ1-N(120)-READ2-3'

The middle section will contain different bases on each molecule.

I planned to amplify these molecules using primers constructed as follows:

5'-P5-READ1-3'
5'-P7-Index-READ2-3'

The final molecules I would send for sequencing should then be:

5'-P5-READ1-N(120)-READ2-INDEX-P7-3'

Will this be a problem? Does this make sense?

- K

[kayleighepps]

Which DNA polymerase is utilised for Illumina sequencing base incorporation?

- K

[rajasereddy]

If each molecule have different sequence then it won't be a problem you can do this. I am not sure which polymerase they use.

[bilyl]

Hi K

Illumina uses same sequence as primer for read 1 and read 2 but the trick is its orientation read 1 primer orientation is exactly opposite to read 2 primer orientation so it won't bind.

When you say sequencing reaction I assume you are mentioning base incorporation if it is so the no the incorporation won't happen >60.

In your case you can use the primers with illumina adapter overhangs to get amplicon product but the problem is it can't be sequenced on illumina machine because of their cluster identification algorithm. You need to have at least random bases for at least first 4 cycles. Better talk to them once.

Cheers
Reddy

That is incorrect. The Read 1 and Read 2/Multiplexing sequencing primers are different but share a common 3' end (~13bp IIRC). The common 13bp end is to create a Y-adapter structure during library prep. The orientation is not "opposite" as you said -- The only case where you would use a primer with the "opposite" orientation is during the Index Read, whose bases are directly upstream of the sequencing primers. This is clearly written in the Customer Sequence Letter, or you can just draw it out yourself.

@Kayleighepps: You should google the "GA Boot Camp" and look at the course that the Broad offers. It's for the older GA system but the principle is practically (almost exactly) the same.