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Old 02-11-2016, 11:57 AM   #1
nutterson
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Default p5/p7 flow cell bound oligo question

i had a question regarding p5/p7 oligos bound to flow cell and wanted to know which of the following was correct in terms of what is actually bound to the flow cell

1) p5 oligo , p7 oligo (sense or antisense only)
2) p5 oligo, p5 oligo complement, p7 oligo, p7 oligo complement

if scenario 1 is correct, then 1/2 of the libraries do NOT bind to p5 flow cell bound oligo since it'd be in the wrong sense. but then do the other 1/2 (that don't bind to p5) bind to p7 making it so that all library sequences are bound?
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Old 02-11-2016, 12:21 PM   #2
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Scenario 1 is correct. Each single strand of DNA has the P7 and P5 oligos attached. They both need to be there for bridge amplification. After bridge amplification, all the reverse strands (attached on their P5 side) are washed off by cleaving of the P5 oligo on the flow cell surface. This leaves all the forward strands in tact for read 1. Read 2 reads the complementary strand, which will be bound on the P5 oligo side (having their P7 oligo sides clipped).
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Old 02-11-2016, 12:32 PM   #3
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thanks for the response.

let's say we have 2 ssDNA as follows:

p5 - insert - p7

complement p5 - complement insert - complement p7

if this is introduced to the flow cell, only 1 of these strands will actually bind flow cell oligo right (either p5 or p7)? meaning 1/2 the template does not bind. however, the non-binding template is necessary pre-sequencing to generate enough template via pcr. is this correct?
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Old 02-11-2016, 01:04 PM   #4
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No, all the ssDNA binds to the flow cell surface (in a perfect world anyhow). The complementary strands from the original dsDNA have the P5 and P7 oligos on opposite sides from one another.

5' - P5 - insert - P7 - 3'
3' - P7 - insert complement - P5 - 5'
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Old 02-11-2016, 01:33 PM   #5
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once the adapters are ligated, wouldn't you (at some point during the following pcr steps) generate strands that are complementary to p5 and p7?
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Old 02-11-2016, 02:25 PM   #6
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Quote:
Originally Posted by nutterson View Post
i had a question regarding p5/p7 oligos bound to flow cell and wanted to know which of the following was correct in terms of what is actually bound to the flow cell

1) p5 oligo , p7 oligo (sense or antisense only)
2) p5 oligo, p5 oligo complement, p7 oligo, p7 oligo complement

if scenario 1 is correct, then 1/2 of the libraries do NOT bind to p5 flow cell bound oligo since it'd be in the wrong sense. but then do the other 1/2 (that don't bind to p5) bind to p7 making it so that all library sequences are bound?
Details of flow cell oligos and some adapter sequences are in the following doc. Please note some portions may be obsolete but should give answer to your question.
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File Type: pdf Flow cell oligos and adapter sequences.pdf (27.5 KB, 171 views)
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Old 02-12-2016, 04:48 AM   #7
kmcarr
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Quote:
Originally Posted by misterc View Post
No, all the ssDNA binds to the flow cell surface (in a perfect world anyhow). The complementary strands from the original dsDNA have the P5 and P7 oligos on opposite sides from one another.

5' - P5 - insert - P7 - 3'
3' - P7 - insert complement - P5 - 5'
No, that is not correct. In the finished, dsDNA library molecules p5 is base paired with the reverse complement of p5 (p5rc) and p7 with the reverse complement of p7. p5 and p7 are not reverse complements of each other. You may be confusing the fact that TruSeq ligates partially non-complementary ('Y') adapters to the dsDNA fragments which do have p5 & p7 at the same, non base paired ends. After PCR enrichment one end of the fragment has p5 and the other p7. Here is a figure showing the initial Y adapters and how enrichment PCR converts those to fully dsDNA library molecules with p7 at one end and p5 at the other.
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File Type: pdf TruSeq_DNA-RNA.pdf (23.5 KB, 124 views)
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Old 02-12-2016, 08:09 AM   #8
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Thank you both. The attached pdf was exactly what I was looking for!

So at the end, the dsDNA can have either p5, p5rc, p7, p7rc attached. Does the flow cell contain seq for p5, p5rc, p7, p7rc as well or only p5, p7 or p5rc, p7rc?
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Old 02-12-2016, 08:21 AM   #9
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Quote:
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Thank you both. The attached pdf was exactly what I was looking for!

So at the end, the dsDNA can have either p5, p5rc, p7, p7rc attached.
It's not really correct to say that the dsDNA "can have either". Every dsDNA library molecule has the one p7 end and one p5 end. Since it is dsDNA these ends will naturally have both the forward and reverse complement of the oligo sequence.

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Does the flow cell contain seq for p5, p5rc, p7, p7rc as well or only p5, p7 or p5rc, p7rc?
The flow cell has only the forward p5 and p7 oligos. Remember that these oligos serve as the primers for bridge amplification so they must be present in the correct 5'->3' orientation; the 5' ends are anchored to the flow cell and the 3' ends are free and extension proceeds from that end during bridge PCR.
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Old 02-12-2016, 08:23 AM   #10
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Gotcha, thank you so much.
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Old 08-09-2017, 08:58 PM   #11
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Originally Posted by kmcarr View Post
It's not really correct to say that the dsDNA "can have either". Every dsDNA library molecule has the one p7 end and one p5 end. Since it is dsDNA these ends will naturally have both the forward and reverse complement of the oligo sequence.



The flow cell has only the forward p5 and p7 oligos. Remember that these oligos serve as the primers for bridge amplification so they must be present in the correct 5'->3' orientation; the 5' ends are anchored to the flow cell and the 3' ends are free and extension proceeds from that end during bridge PCR.
The both of complement ssDNA can hybirdize to p5 and p7 oligos on flow cell. But in the skematic of Illumina, only p7 oligos can extand to be the initial template before bridge PCR. So Why p5 can't extand?
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