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Old 10-19-2011, 01:12 PM   #1
skblazer
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Default Does non-Y-shaped adapter work for Illumina library?

I want to add inline barcode within custom adapters for multiplexing sequencing. The sequencing lab manager suggested me just adding the 4-6bp indexes on one end of adapter. Thus two oligos are not complimentary on the end to form a Y-shape any more.

She explained to me that they didn't anneal them and just mixed equimolar amounts for ligation. The one with 5'Phos goes on 3'-end of library fragment. The non-phosphorylated one goes on 5'-end of fragment.

I don't know if someone here did this before for a regular paired-end library? But I can't figure out how the DNA ligase works on this condition.
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Old 10-20-2011, 08:09 AM   #2
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You can do that by not using Y adapters. Instead use 2 adapters. An "F" and an "R" adapter (or whatever you want to call them). The down side is that 1/2 of your recombinant molecules will have "F" on both sides or "R" on both sides.

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Old 10-20-2011, 10:00 AM   #3
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Thanks Phillip, and I can't filter them out through the PCR enrichment step?

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Originally Posted by pmiguel View Post
You can do that by not using Y adapters. Instead use 2 adapters. An "F" and an "R" adapter (or whatever you want to call them). The down side is that 1/2 of your recombinant molecules will have "F" on both sides or "R" on both sides.

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Old 10-21-2011, 10:25 AM   #4
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Sure, PCR suppression should result in preferential amplification of the amplicons with different adapters. It will work fine as long as you get the adapter sequences right.

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Old 10-22-2011, 07:05 PM   #5
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I'm always feeling stupid about this, but how do the Y-shaped adapters guarantee getting fragments with one A and one B adapter? From the ligase's point-of-view, how does one end of dsDNA look different from the other end? Or is it that each Y adapter ends up ligating one strand to one end of a fragment and the other strand to the other end, as the kinetics are set to favor pseudo-circularization?
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Old 10-23-2011, 07:47 AM   #6
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I think it is a single Y adapter, not two different Y adapters, in the reaction.
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Old 10-23-2011, 01:09 PM   #7
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Quote:
Originally Posted by krobison View Post
I'm always feeling stupid about this, but how do the Y-shaped adapters guarantee getting fragments with one A and one B adapter? From the ligase's point-of-view, how does one end of dsDNA look different from the other end? Or is it that each Y adapter ends up ligating one strand to one end of a fragment and the other strand to the other end, as the kinetics are set to favor pseudo-circularization?
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I think it is a single Y adapter, not two different Y adapters, in the reaction.
Correct (if I understand this correctly). There is ONE Y-shaped adapter. The adapter is Y-shaped because the outer portions are not complementary to each other. However, the inner portions are. The outer portions not being complementary allow different primers to sit down on each strand. If they were complementary (ie, one set of linear adapters), then the primers would simply anneal together as the primers themselves would be perfectly complementary. Since the inner portions are complementary, they anneal together and are able to ligate onto the A-tailed fragments. As the 5' end of each strand gets an A-added on, the strand of the Y-shaped adapter that ligates onto the 5' end will be on one side of the fragment on one end and the other side on the other end. The end result being it's set up perfectly for PCR when the strands are denatured.
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Old 10-23-2011, 06:41 PM   #8
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Heisman is pretty much dead on....(but A-addition is on the 3'-ends of each strand).

It helps to think in terms of individual strands (rather than ends of the dsDNA molecule). Each strand gets a different branch of the Y ligated to it. This post has a detailed diagram.
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Old 10-24-2011, 04:26 AM   #9
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The critical factor here is that T4 DNA ligase will only link a 5' end to a 3' end and will only do so when those ends are situated in an annealed double strand.

The structure of DNA takes care of the rest: if the top strand of the Y adapter terminates in a 3' end, the bottom strand must be a 5' terminus. On the other end of the insert, then, the top strand of the adapter must terminate in a 3' end to be ligated.

The 5' strand of the Y adapter and the 3' strand of the Y adapter are different adapters, one "F" and one "R". So after PCR, as long as adapter was ligated to both ends of the insert, it will have "F" on end and "R" on the other.

By the way, the "T" overhang is gravy is this scheme. It would work exactly the same with blunt adapters and blunt inserts. But adapter-adapter ligation would be rampant (as well as insert-insert.)

Elegant. In the early days of the SOLiD I spent long hours pouring over the fragment library creation protocol trying to find a way to avoid those F-insert-F and R-insert-R molecules that would consume 1/2 the constructs created during ligation. But I could only imagine this happening via circularization. But the very basis of the ligation of dsDNA provided another method that never occurred to me. (Until someone showed me the Y-adapter scheme.)

But since both Illumina and SOLiD library construction protocols call for enrichment PCR, it doesn't really matter that much. That 2x difference is a single cycle of PCR. But given the choice, why not opt for a 2x advantage initially?

More mysterious is that in recent times Illumina apparently has been instructing their FAS's to treat the Y-adapters as "secret". While I am totally opposed to such secrecy on general principle -- as scientists I think we should just ban it entirely -- I can't imagine a possible purpose for pretending like something that is well know, is a secret. The novices will not find out from Illumina staff. But as soon as they peruse the literature they will know. Competitors will, of course, be aware of it. So who benefits from this deliberate obfuscation?

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Old 10-24-2011, 06:36 AM   #10
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Hi Phillip,

You means it doesn't work if I didn't anneal two oligos? But in the first reply you said I can obtain 50% recombinant moleculars of F-insert-R?

I'm sorry I'm confused with this. So, T4 DNA ligase cannot align two single-strand oligos to a double-strand DNA molecular?

Quote:
Originally Posted by pmiguel View Post
The critical factor here is that T4 DNA ligase will only link a 5' end to a 3' end and will only do so when those ends are situated in an annealed double strand.

The structure of DNA takes care of the rest: if the top strand of the Y adapter terminates in a 3' end, the bottom strand must be a 5' terminus. On the other end of the insert, then, the top strand of the adapter must terminate in a 3' end to be ligated.

The 5' strand of the Y adapter and the 3' strand of the Y adapter are different adapters, one "F" and one "R". So after PCR, as long as adapter was ligated to both ends of the insert, it will have "F" on end and "R" on the other.

By the way, the "T" overhang is gravy is this scheme. It would work exactly the same with blunt adapters and blunt inserts. But adapter-adapter ligation would be rampant (as well as insert-insert.)

Elegant. In the early days of the SOLiD I spent long hours pouring over the fragment library creation protocol trying to find a way to avoid those F-insert-F and R-insert-R molecules that would consume 1/2 the constructs created during ligation. But I could only imagine this happening via circularization. But the very basis of the ligation of dsDNA provided another method that never occurred to me. (Until someone showed me the Y-adapter scheme.)

But since both Illumina and SOLiD library construction protocols call for enrichment PCR, it doesn't really matter that much. That 2x difference is a single cycle of PCR. But given the choice, why not opt for a 2x advantage initially?

More mysterious is that in recent times Illumina apparently has been instructing their FAS's to treat the Y-adapters as "secret". While I am totally opposed to such secrecy on general principle -- as scientists I think we should just ban it entirely -- I can't imagine a possible purpose for pretending like something that is well know, is a secret. The novices will not find out from Illumina staff. But as soon as they peruse the literature they will know. Competitors will, of course, be aware of it. So who benefits from this deliberate obfuscation?

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Old 10-24-2011, 07:38 AM   #11
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Ah, I thought it would be understood that your adapters have to be double stranded to be ligated to your insert with T4 DNA ligase.

If you want to use separate F and R adapters, that is okay. But you need to order two oligos for each adapter, then. A top and bottom strand for each. Also, you would want to include modifications to prevent adapter-adapter ligations from occurring.

Anyway, I was not providing a protocol, just answering your question as to whether it was possible to use non-Y adapters for Illumina libraries. There will be various pitfalls to be avoided, etc.

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Old 10-26-2011, 05:50 PM   #12
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Many thanks, Phillip. This is very helpful.
Quote:
Originally Posted by pmiguel View Post
Ah, I thought it would be understood that your adapters have to be double stranded to be ligated to your insert with T4 DNA ligase.

If you want to use separate F and R adapters, that is okay. But you need to order two oligos for each adapter, then. A top and bottom strand for each. Also, you would want to include modifications to prevent adapter-adapter ligations from occurring.

Anyway, I was not providing a protocol, just answering your question as to whether it was possible to use non-Y adapters for Illumina libraries. There will be various pitfalls to be avoided, etc.

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Old 10-27-2011, 06:06 PM   #13
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Merci -- now I see! The "think about it in terms of strands" is the real kicker -- a given strand can only get one of each.
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