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Old 01-26-2011, 11:55 PM   #1
bhennuy
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Default DSN normalization with TruSeq kits

Hi,
Has anyone already try the DSN normalization with the new RNA TruSeq kits ?
How to fragment the total RNA and to link this step with the Truseq protocol?
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Old 02-18-2011, 12:39 AM   #2
QTLdum
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Default Start with the RFP

As far as I can see from the TruSeq application note, it should be possible to enter the protocol at the "Incubate RFP" step after adding the Elute, Prime, Fragment mix. The mix should perform the fragmentation step.

My question for the DSN procedure with TruSeq is on the primers used for the final amplification steps after the DSN treatment. The Illumina application note for DSN indicates using the PCR Primer PE 1.0 and PE 2.0 from the old RNA sample prep kits. However, I assume these will not work with the subsequent TruSeq cluster generation and sequencing steps. Does any one have an idea on the sequence of the primers required for DSN in combination with the TruSeq kits?
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Old 04-01-2011, 01:09 PM   #3
Hisequser
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We got it to amplify, but we are waiting for the sequence data to see how well the protocol performed.
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Old 04-04-2011, 10:50 PM   #4
Katie1
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This should answer the primer question provided by Illumina Tech Support. My question is still where to step in with DSN treatment with TruSeq kit. As the old protocol advices to start DSN with already prepared library.

If user select to use Truseq RNA sample prep kit, then they need to use
PCR primer cocktail (PPC) from the Truseq RNA sample prep kit for the enrichment step in the DSN normalization protocol. The PE PCR primers
are different from the Truseq PCR primers, so customer CANNOT use the
Illumina 100 RXN Paired End DNA Sample Prep Oligo Only Kit, catalog #
PE?102?1003 (100 samples).
However, for the Truseq RNA sample prep kit, there is no oligo only kit available to order yet (will be released sometime this year), so customer has to use PCR prime cocktiail from the Truseq kit.
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Old 04-05-2011, 06:53 AM   #5
Katie1
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Answering to myself

DSN step should be carried out after adapter ligation. Still there is a risk that DSN kit does not work perfectly with TruSeq because of the longer adapters which are needed for multiplexing indexes. Also there is a risk to loose the input as the starting material is very low.
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Old 04-08-2011, 09:13 AM   #6
Hisequser
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Quote:
Originally Posted by Katie1 View Post
Answering to myself

DSN step should be carried out after adapter ligation. Still there is a risk that DSN kit does not work perfectly with TruSeq because of the longer adapters which are needed for multiplexing indexes. Also there is a risk to loose the input as the starting material is very low.
Hi Katie, Why should the DSN step carried out after adapter ligation? We have tried DSN treatment on a Truseq RNA prep and got a pretty decent library (used Truseq Primer cocktail). Still waiting on the data to see how it compares to the pre-DSN sample.
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Old 05-23-2011, 10:40 AM   #7
clinnen
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Hi Hisequser,
At which step in the truseq protocol did you carry out the DSN treatment? Also, any update on your results?
Thanks!
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Old 05-23-2011, 10:50 AM   #8
Hisequser
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Hello Clinnen,

Based on our informatics results and a few discussions with Illumina experts, the Truseq protocol does not perform well with DSN (Although we did obtain a pretty good size library after DSN treatment), the number of unique reads was low.. We repeated the same sample using traditional RNA protocol but skipping polyA selection, it will be sequenced soon. Will try to keep you posted.
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Old 05-31-2011, 08:14 AM   #9
planseq
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Hi Hisequser,

Could you comment on the cause of this incompatibility of the Truseq protocol with the DSN normalization? Is it the length of the adaptors that is interfering, or do you think there is some other reason that it didn't work well?
Thanks!
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Old 05-31-2011, 09:31 AM   #10
mnkyboy
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I have seen little difference using DSN on TruSeq libraries. I just use the TruSeq PCR primer and master mixes with those conditions.

DSN is a pain and inconsistent but the DSN enzyme should work on any completed library as long as you have the correct primer sequences for the adapters.
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Old 05-31-2011, 11:17 AM   #11
Katie1
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Hi,

DSN did not work in our case. I started the DSN prtocol with 100 ng adapter ligated and PCR enriched library samples as Illumina Tech suggested. Unfortunatelly the final concentration in qubit was undetectable and we did not preceed sequending with those samples.

Hope next time would work better but dont know how.
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Old 01-26-2012, 06:14 AM   #12
whw
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Hoping to get this thread going again–

I would like to use DSN on multiplexed TruSeq libraries but I'm concerned about compatibility. Illumina does not support DSN with the TruSeq kits because of unspecified trouble with the adaptors–they cite adaptor length as an issue but are short on specifics. I originally thought that long adaptors would hybridize and lead to degradation of all species but a colleague recently tried digesting a TruSeq library and found no reduction in abundant species. Is it possible that the forked adaptors are actually preventing DSN from attacking any transcripts?

Does anyone have experience with DSN digestion on TruSeq libraries? I'd be interested to hear about any negative results or any successes or if there is an alternate barcoding system that works with DSN.
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Old 01-26-2012, 09:55 AM   #13
mnkyboy
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It does not work. I have learned this first hand. This is Illumina's official reason:

Quote:
Can the TruSeq RNA libraries be used with the DSN Normalization protocol?
The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:
The Elute-Prime-Fragment mixture is not optimized for non-poly(A) RNA.
The PCR primers in TruSeq RNA were optimized for a high yield output. The DSN provides much lower yield due to the duplex-specific nuclease activity, so the PCR primers are not efficient to amplify the DSN-treated TruSeq RNA libraries.
Why are you wanting to use DSN?
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Old 01-26-2012, 10:43 AM   #14
whw
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I've heard that from Illumina as well but I'm not really satisfied with the answer. The issue during EPF is probably due to the use of oligo-dT beads to bind and clean up the reaction. I would guess that DSN treatment might degrade the poly-A tails, preventing the use of beads. However if you use a different isolation method, say a column based system, there's no reason it shouldn't work. As for the primer efficiencies I don't see any reason why you couldn't adjust the concentration for a smaller amount of sample.

However! Maybe you tried all those things and they didn't work, what was your experience with DSN?

We are interested in low abundance transcripts in lymph node tissue. Coverage is very important since we are essentially looking for needles in an immune cell haystack. I am hoping that DSN will improve our chances.
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Old 01-26-2012, 10:51 AM   #15
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We were usind DSN to remove high abundance transcripts, mainly rRNA and have switched to the RiboZero beads. I am sure with some time you could get DSN to work but we did not want to spend that since our solution was much easier with the beads. DSN is very finicky but we had good success using the older Illumina library kits with it, but then you lose the ability to multiplex. You may have to use some custom primers for increased yield.
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Old 02-12-2012, 12:43 PM   #16
pmiguel
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Quote:
Originally Posted by whw View Post
I originally thought that long adaptors would hybridize and lead to degradation of all species but a colleague recently tried digesting a TruSeq library and found no reduction in abundant species. Is it possible that the forked adaptors are actually preventing DSN from attacking any transcripts?
Seems more likely that the DSN was swamped with duplexes derived from adapter-adapter annealing (just as you initially posit).

Might work to perform normalization on the cDNA pre-ligation. Then re-synthesizing 2nd strands. Then blunting/ligation.

The problem there would be that reverse transcription in the TruSeq is designed to work on a much lower amount of RNA (after removal of rRNA). So there might not be enough reagent to convert all that additional RNA.

I guess you could generate cDNA using some other kit. Normalize, then enter TruSeq construction. At that point you might as well use the cheaper DNA TruSeq kit, though.

--
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Old 03-12-2012, 10:02 PM   #17
sterakura
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I tired DSN-Library from total RNA with TruSeq RNA Sample Prep Kit ver.2.
Analysis of Agilent Bioanalyzer showed that DSN-Library contained abnormal peaks like ladders.
I've never seen this abnormal ladder-like peaks in DSN-Library prepared with older mRNA-Seq Kit.
Have you seen abnormal ladder-like peaks in DSN-Lib prep with TruSeq Kit?
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Old 03-13-2012, 07:02 AM   #18
pmiguel
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The ladder peaks may be from the control DNAs that are an optional part of the TruSeq kits. Did you use the control DNAs during library construction?

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Old 03-13-2012, 07:08 PM   #19
sterakura
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Hi, Phillip

I considered the possibility of TruSeq's in-line-control contamination,
but obtained reads were not aligned on sequence of in-line control DNA.

I think that TruSeq adaptor could cause the ladder peaks artificially.
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Old 04-12-2012, 06:06 AM   #20
led_o_zep
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Maybe I'm missing something obvious, but if the issues are with the adapters, then why not DSN treat the cDNA before end-repair, etc? -nevermind, I missed the bit about the cDNA synth being optimized for low-input poly-a, etc.

Last edited by led_o_zep; 04-12-2012 at 06:35 AM. Reason: reread earlier messages
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