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Old 06-27-2011, 12:36 AM   #81
nbogard
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Why is the phosphorothioate modification (in the adapter) at the 3'-end that is proximal to the ligation? Shouldn't that 3'-end be protected after the ligation? I don't understand why the modification is not added to the Y-end of the opposite adapter. It seems that the 3'-end of only that adapter would be vulnerable to the nuclease.

Also, do locked nucleic acids (as used in the Tufts protocol) serve the same purpose: to prevent 3' to 5' nuclease activity? Do either the phosphorothioate bond or locked nucleic acids aid in the annealing of the two adapters to each other?

Any clarification or insight would be greatly appreciated. Thanks much.
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Old 06-27-2011, 01:11 AM   #82
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Hi nbogard,

Exonucleases in the ligase prep can chew off the adaptor "A" overhang. Two of these blunt-end ligated together will amplify nicely. While the probability of exonuclease activity and frequency of blunt end ligation is small, the adaptor is in great excess over DNA library and will amplify later more efficiently (due to being shorter than the library).

LNAs will increase the apparent Tm, but may hinder ligation. I'm not sure if the ligase will accommodate bulky LNA backbones. You'll have to test... anyone with experience here?
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Old 06-27-2011, 01:44 AM   #83
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Thanks sci_guy!

Is the exonuclease activity in the ligation prep from leftover T4 DNA Pol used in the earlier end-repair reaction? If the contamination is significant, that would explain the reason for the phosphorothioate modification at the ligation end. But why the lack of the modification on the opposite adapter that also contains an exposed, single-strand 3'-end?
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Old 06-27-2011, 02:31 AM   #84
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Could be leftover activity. But I would assume that the chaotropic salts and EtOH in the column would kill the activity.

Some degradation of the 3' end in the Y-tail is not a problem. The PCR primer will still anneal nicely. However, annealing of two blunt-ended Y adaptors yields an amplifiable product.

Have a look at this thread: http://seqanswers.com/forums/archive...hp/t-1765.html
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Old 06-27-2011, 04:28 PM   #85
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Thanks again, sci_guy. Makes sense.

All the best!
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Old 07-07-2011, 07:49 AM   #86
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Default Illumina sequence manipulations

Hello, new to NGS, I've found this site extremely helpful. For my own edification and to help others in the same fog I was in, I put together a document with all the manipulations involved in single-end and multiplexed library preparation. Information from various posts on this site was critical in being able to do this. I hope that people can check this document for errors, both in overall structure of the scheme, and in the specific oligo sequences.
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File Type: pdf Illumina sequence manipulations.pdf (21.2 KB, 934 views)
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Old 07-07-2011, 08:33 PM   #87
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Hi, frc1230! Your schematic is great. Thanks for the contribution.

-Nick
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Old 07-14-2011, 09:54 AM   #88
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Default Homemade TruSeq adapters

Hi all, for various reason I am generating homemade TruSeq adapters. After annealling the universal and index oligos, they run at ~65bp on a 2.5% gel as I expected. However, Illumina TruSeq adapters from the kit, run as a positive control, were at ~100bp. Anyone have any experience of why this is (given they are supposed to be ~65bp)? Is it anything to do with the Y structure and does this, presumably, mean that my homemade adapters have not annealed properly? If anything I would have thought the Y-structure would have meant they run smaller than 60bp, not larger.

Thanks
nb. To anneal I mixed eqimolar amounts of index and univeral oligos in 50mM NaCL, incubated at 95C for 5 min and allowed to cool to RT in the heat block (2hr). The oligos are modifed 5'phosphate and 3'phosphorothioate respectively.
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Old 09-02-2011, 06:22 AM   #89
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frc1230 -> thanks for the nice schema!
for improving next version: it might be misleading when you write qPCR (from Quail et al) primers below multiplexing sequences, because they will work with only some of the indexes (last base of primer is first base of index)
otherwise very nice
best,
Lukasz
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Old 09-28-2011, 04:58 PM   #90
frc1230
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Default TruSeq sequence manipulations for library preparation

Kulukulas, thanks for the correction on the oligos.

The attached document contains my best guess as to how the new TruSeq kit works: by a similar principle as in my previous schematic, but there's one critical difference. By the time adapter ligation is complete, the material is cluster-formation-ready. The PCR amplification is solely to amplify the material, it does not add any sequences (unlike in the previous procedure, where the PCR amplification added the 'right-hand' sequence required for cluster formation). (Illumina Tech support was willing to confirm this statement, but they do not provide the sequences of the PCR oligos. It is nevertheless reasonably clear what those sequences must be like).
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File Type: pdf Illumina TruSeq sequence manipulations.pdf (15.0 KB, 571 views)
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Old 09-29-2011, 03:12 AM   #91
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Quote:
Originally Posted by frc1230 View Post
Kulukulas, thanks for the correction on the oligos.

The attached document contains my best guess as to how the new TruSeq kit works: by a similar principle as in my previous schematic, but there's one critical difference. By the time adapter ligation is complete, the material is cluster-formation-ready. The PCR amplification is solely to amplify the material, it does not add any sequences (unlike in the previous procedure, where the PCR amplification added the 'right-hand' sequence required for cluster formation). (Illumina Tech support was willing to confirm this statement, but they do not provide the sequences of the PCR oligos. It is nevertheless reasonably clear what those sequences must be like).
Thanks for posting this it's really helpful and is something I'd been trying to fathom out myself recently - H
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Old 11-23-2011, 03:12 PM   #92
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Quote:
Originally Posted by frc1230 View Post
Kulukulas, thanks for the correction on the oligos.

The attached document contains my best guess as to how the new TruSeq kit works: by a similar principle as in my previous schematic, but there's one critical difference. By the time adapter ligation is complete, the material is cluster-formation-ready. The PCR amplification is solely to amplify the material, it does not add any sequences (unlike in the previous procedure, where the PCR amplification added the 'right-hand' sequence required for cluster formation). (Illumina Tech support was willing to confirm this statement, but they do not provide the sequences of the PCR oligos. It is nevertheless reasonably clear what those sequences must be like).

thanks a lot!! makes things so much clearer!
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Old 01-01-2012, 05:22 AM   #93
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Hi,
Is there a chance that the sheared genomic DNA fragment ligate with both adapter 1(or 2)
at both ends? Or it's not a problem later on.
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Old 02-09-2012, 01:37 PM   #94
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There is only one adaptor; it has a “Y” configuration with a region of complementary sequence at the end that ligates to the genomic DNA, and two different tails that correspond to the two different primers. This design should yield two different ends after PCR amplification.

My question…Illumina’s PCR oligos extend into the region of complementary sequence which could give rise, be it with low efficiency, to products that have two A ends or two B ends. I’m not familiar with a great deal of paired end sequencing results, but how often does this cause a problem (failed sequences, or reads without paired reads)?

The TUFTS design moves the priming back to exclude the complementary region, which eliminates the possibility of products with identical ends (well at least identical ends due to the adapter/PCR design). Has anyone tried modifying the Illumina PCR oligos to remove the region of complementary sequence or is this not a problem?
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Old 02-12-2012, 12:20 PM   #95
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During ligation the only possible product would have A-insert-B once strand-denatured. You seem to be presuming that the TruSeq primers (from the "PPC" tube) span the entirety of the adapters.

(1) Illumina, as far as I know, refuses to disclose the sequence, or any other information about the composition of their TruSeq enrichment PCR primers. Have you somehow determined their sequence? Eg, with a limited Snake Venom Diesterase digestion followed by Mass Spec, or some other method.

(2) Illumina does specify, if asked, that the PPC primers are compatible with all TruSeq indexes. Which makes it likely that they do not span the entirety of the adapters. Otherwise PPCs would likely be index specific.

Thus it seems unlikely that the TruSeq PPC primers include sequence in the insert-proximal (complementary) region.

--
Phillip
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Old 02-13-2012, 01:03 PM   #96
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Phillip,
Thanks for the reply. Sorry, my post lacked specificity; my question is more of a dinosaur at this point. I was wondering about the original PE adapter/primers listed at the beginning of this thread. You are correct about the TruSeq design; it doesn’t make sense that the primers would span the entire adapter like the original PE adapter design.
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Old 03-05-2012, 07:49 AM   #97
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Very very much thanks
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Old 03-20-2012, 07:53 PM   #98
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Hi,
Illumina company has updated the small RNA cloning kit in 2010, known as TruSeq™ Small RNA*Sample*Preparation kit, so I wonder if the sequences of the RNA adapters and primers have beed changed. Additionally, because the manual does not provide the sequence of each linker or primer, can you kindly provide their exact sequence, expecially the 5' RNA Adapter (RA3), 3' RNA Adapter (RA5) and index containing RNA PCR primer (anyone of 48 primers)?
Thank you very much!
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Old 03-29-2012, 01:19 AM   #99
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Hi. Is it the old PE adapters supplied in Paired End Sample Prep Kit contains a mixture of 2 types of double stranded adapters?

While the new TruSeq DNA sample prep kit only comes with one Y-adapter?

If yes for the 1st question, does it mean there is a possibility that the fragments will probably ligated with 2 same adapter at both ends, and also 2 different adapters at both ends, and only fragments with both different adapters are qualified for cluster generation?

Thank you.
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Old 03-29-2012, 01:38 AM   #100
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Quote:
Originally Posted by Akira View Post
Hi. Is it the old PE adapters supplied in Paired End Sample Prep Kit contains a mixture of 2 types of double stranded adapters?

While the new TruSeq DNA sample prep kit only comes with one Y-adapter?

If yes for the 1st question, does it mean there is a possibility that the fragments will probably ligated with 2 same adapter at both ends, and also 2 different adapters at both ends, and only fragments with both different adapters are qualified for cluster generation?

Thank you.
1st Q - old adapters
No it is the same Y shaped adapter: the P7 has additional sequence to incorporate 2nd read sequencing primer and annealing portion for flowcell so that clustering for the second read can be performed. Also the amplification primers are different for PE libraries to reflect the changes in the P7 sequence. I have attached a doc I give to students/interested parties to illustrate the adapter structure.

2nd Q - Truseq
TruSeq adapter is a complete adapter ie it technically does not require any amplification step as all the sequences required to anneal to the flowcell are included in the adapter - the old adapters absolutely required amplification otherwise the portion that anneals to the flowcell would not be present in the library.
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File Type: pdf SR_&_PE_Adapters_&_Primers.pdf (352.6 KB, 363 views)
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