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Old 08-22-2012, 03:05 PM   #1
jtackney
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Default Ion Plus Fragment Library Prep Efficiency

I've had some experience with both Illumina library preps and Ion Torrent library preps and I am thinking about efficiencies on a basic molecular level. Ion Torrent/454 preps use blunt end ligation of 2 different adapters, while Illumina uses TA-ligation of 1 adapter. Correct the following logic if I'm wrong, if we have 4 dsDNA molecules:

Illumina's protocol should arrive at 8 effective ssDNA molecules, for 100% efficiency. The Y-adapter prevents misdirectional adapter ligation, but allows adapter-adapter ligation dimers to still form.

Ion Torrent's protocol should arrive at 2 effective dsDNA molecules, for 50% efficiency, as 25% of the fragments will have A-fragment-A and 25% will have P1-fragment-P1. Granted, these will be non-amplifiable but they do sequester true DNA fragments in solution. Additionally, the blunt-end ligation protocol would hypothetically also cause a whole mess of chimeric ligation products, such as A-fragment-fragment-P1. Fragments shouldn't ligate on both sides of an adapter due to the double T tailing, which also prevents misdirectional adapter ligation. Adapter ligation dimers will still form, at much the same rate as Illumina adapters (?), though composed of 3 combinations of the 2 adapters.

Any issues with this? Why didn't Life Tech go down the TA route as that would solve a lot of the blunt-end problems? And yes, I am aware that there are some groups that have developed their own Y adapters for use on the Ion Torrent/454.

Looking forward to comments and/or kind remarks that someone else has already talked about this
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Old 08-22-2012, 04:33 PM   #2
RCJK
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Just an FYI...454 also uses Y adaptors similar to Illumina. This has been the case for the past few years since the release of their Rapid Library prep kits.
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Old 08-22-2012, 10:36 PM   #3
genseq
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Cool

Quote:
Originally Posted by jtackney View Post
The Y-adapter prevents misdirectional adapter ligation, but allows adapter-adapter ligation dimers to still form.

Additionally, the blunt-end ligation protocol would hypothetically also cause a whole mess of chimeric ligation products, such as A-fragment-fragment-P1. Fragments shouldn't ligate on both sides of an adapter due to the double T tailing, which also prevents misdirectional adapter ligation. Adapter ligation dimers will still form, at much the same rate as Illumina adapters (?), though composed of 3 combinations of the 2 adapters.
Y-adapters can not form dimers due to a T-tailing.
A-B (P1) can not form dimers due to the lack of 5'-P.
The blunt-end ligation "fragment-fragment" is rare because of the high concentration of adapters.
Attached Images
File Type: jpg Illumina Y-adaptors.JPG (9.2 KB, 92 views)
File Type: jpg Ti-AB-Y.JPG (51.1 KB, 129 views)
File Type: jpg AB-Y.JPG (30.6 KB, 104 views)
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Old 08-23-2012, 02:14 PM   #4
jtackney
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Thanks RCJK. I had forgotten that about the 454 quick prep changes.

And thank you genseq for the images. I had seen one of those before, which prompted my initial post. I do recognize that the adapters *shouldn't* form self ligating dimers, for the reasons you state, but they do in some preps so it's interesting why.... I would think the Y adapters form them much less readily, but I guess it comes down to the thermodynamics of the reaction.

Regardless, it does seem like Y adapter-based chemistry is more efficient.
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Old 08-24-2012, 12:42 PM   #5
snetmcom
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sticky adapters are more efficient. I don't think anyone debates that. Blunt end ligation is just a little faster and cheaper as it doesn't require tailing. You can make libraries from pg of material with blunt end ligation, so I'm not sure if the efficiency is really an issue. Blunt is theoretically 50% less efficient if you assume perfect tailing and ligation for sticky adapters. That's probably a cycle or so change in amplification.
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Old 09-23-2012, 11:19 PM   #6
Vaida
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Has anybody experience to create Y adapters for sequencing with IT?
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Old 09-25-2012, 10:52 PM   #7
genseq
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Quote:
Originally Posted by Vaida View Post
Has anybody experience to create Y adapters for sequencing with IT?
Hi, Vaida.

I have not experience with Y adapters, but can to calculate their sequences for your.
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Old 06-14-2013, 05:51 PM   #8
seqfan05
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Default design our own P1/A ion torrent adapters

Quote:
Originally Posted by Vaida View Post
Has anybody experience to create Y adapters for sequencing with IT?
did anybody have any experience with the disign of Y-adapter sequeces for Iontorrrent? besides. I was wondering if it's possible to redesign my own IT P1/A1 adapter sequence based on the initial iontorrent A/P1 adapter sequences. since I want to perfrom multiplexing sequence on 318 chip, the barcode sequences kit provide by lifetech is too expensive to be offordable.
best regards
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Old 07-17-2013, 05:38 PM   #9
vandy
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I am new to sequencing world. We are trying to perform library preparation for exome sequencing using Ion Proton. During the process tehre is three days hybridization step which got interrupted due to some power failure overnight.

We are in dilemma that should we continue with hybridization step or should be abort and start again with the new sample.

Has anyone had this issue before?

Can you suggest , whats the best possibility?

Thanks in advance for all your suggestions.

Regards

vandy
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Old 07-22-2013, 02:38 PM   #10
JackQuist
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Why didn't Life Tech go down the TA route as that would solve a lot of the blunt-end problems?

The reason is Illumina owns the IP for that form of library prep.
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Old 07-23-2013, 01:31 PM   #11
snetmcom
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Quote:
Originally Posted by JackQuist View Post
Why didn't Life Tech go down the TA route as that would solve a lot of the blunt-end problems?

The reason is Illumina owns the IP for that form of library prep.
They do not own an IP for tA libraries.
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Old 09-11-2020, 07:58 AM   #12
kevin zhang
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can you re-send this article?
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Old 09-12-2020, 04:30 AM   #13
BioDynami
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This is the design of BioDynami NGS DNA Library Prep Kit (Ion Torrent Platform): the insert concatemer ligation (due to blunt end ligation) is eliminated with our unique technology.



more details at https://biodynami.com/product/ngs-dn...-prep-kit-ion/
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