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Old 05-13-2011, 03:10 AM   #1
Sakana
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Default Why Can't SureSelect work in TruSeq

Hi. I'm a member of a experimental reagent company. I want to know

"Why Can't SureSelect work in TruSeq" .

Please tell me about this.

Thanks in advance.
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Old 05-13-2011, 06:48 AM   #2
krobison
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A key bit of the SureSelect kits are blocking oligos -- it is critical in these procedures to prevent library molecules from "daisy chaining" -- hybridizing through complementarity of their adapter sequences. Such chaining will result in pull-down of off target fragments.

The blocking oligos are complementary to the adapter sequences. Hence, any change in the library oligos will lead to incompatibility / poor performance.
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Old 05-13-2011, 12:19 PM   #3
GW_OK
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It can also depend on what read length you are using. Sureselect capture is designed for ~150bp insert size, while the new Truseq reagents are designed for longer read lengths. You don't want to be doing PE100 reads on a 150bp insert sample size. The Truseq capture reagents are designed to take longer read lengths into account.
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Old 05-14-2011, 01:47 AM   #4
Sakana
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Thank you for your easily comprehensive answer. I got it.

Therfore Agilent can't sell SureSelect for Illumina Platform unless they develop new product for it.
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Old 05-16-2011, 10:01 AM   #5
NextGenSeq
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Even for Roche you have to synthesize the blocking oligos for TruSeq. They have the sequences listed on the Nimblegen web site for the blocking oligos.
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Old 08-23-2011, 09:57 PM   #6
Heisman
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Quote:
Originally Posted by GW_OK View Post
It can also depend on what read length you are using. Sureselect capture is designed for ~150bp insert size, while the new Truseq reagents are designed for longer read lengths. You don't want to be doing PE100 reads on a 150bp insert sample size. The Truseq capture reagents are designed to take longer read lengths into account.
Do you (or anyone) know how reagents would be changed simply for longer insert sizes?

If the full reason for why they aren't compatible is simply the blocking oligos, could you synthesize your own blocking oligos and then have SureSelect be compatible with TruSeq? Has anybody tried this?
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Old 08-23-2011, 10:13 PM   #7
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In my experience, the only reason this wouldn't work is that you n eed the correct blocking oligos. Length of the pull down in this range and higher doesn't seem to be much of a factor.
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Old 10-12-2011, 08:53 AM   #8
brentonm
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Default Agilent Sureselect and Truseq

I've done a few dozen Sureselect captures with the Illumina Truseq Prep. My on-target sequencing suffers quite a bit. My on target % went from 60-70% to 30%, unfortunately, but it still worked. I used the standard hybridization protocol without any different blocking primers, like what is recommended by Roche. For my next hybridization, I'll try the Roche blocking primers and see if it helps my on target capture.
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Old 05-10-2012, 12:31 PM   #9
YW0712
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Hi All,

Thanks for all the info. I was just wondering for the SureSelect capture following TruSeq library prep, do you still use all the SureSelect blocking oligos (#1-3), in addition to the ones specific for the TruSeq adaptors? I'm thinking that I might be able to leave out the blocks specific to their adaptors (instead of the TruSeq ones).

Thanks in advance for any insights!
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Old 05-10-2012, 01:52 PM   #10
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I've done this recently and it can be modified to work great with TruSeq libraries.

First....Read this protocol in detail and make sure you understand every step. http://openwetware.org/wiki/Image:Geller_exome.pdf It's great. I made a small modification in that this paper uses blocking oligos that will immediately anneal to each other...not really a great strategy. I'm just blocking one strand on each adapter...eliminating any complementarity between library molecules.

What I've done...leave out Agilent's block #3 (the 0.6ul per capture), and add the following mixtures of blocking oligos (all at 50uM).

With the modified blocking oligos the PCT_SELECTED_BASES was 73%...which is within range of normal SureSelect in our hands (here our positive control (stock sureselect) was 80%.

I'm still working on the split blocking strategy as it's an unnecessary workflow complication to have an index specific block. I was not very surprised that this split adapter strategy didn't perform well as the Tm's are too low to be very stable in the hyb, but it was worth a shot. My second attempt will fix this issue I hope!

Anyway I hope this helps....let me know if you have any questions...hopefully I didn't make any mistakes or typos below!

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File Type: pdf SureSelect-TruSeq Adapter Strategy.pdf (64.7 KB, 391 views)
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Old 05-10-2012, 04:22 PM   #11
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Hi Eco,

Thanks for posting your results of your experiment testing various blocking oligo strategies. Have you tried using a generic oligo with deoxyinosine substituted for all six variable nucleotides of the blocking index oligo? I've been using this approach with a custom NimbleGen sequence capture experiment. The PCT_SELECTED_BASES is usually 55-65%. It's not as good as specific blocking oligos but good enough for my needs.

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Old 05-10-2012, 04:40 PM   #12
ECO
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AA, I haven't tried that, that's a great suggestion. My next approach is to increase the Tm of the split blockers by using modified bases (2MO and LNA).
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Old 05-10-2012, 05:06 PM   #13
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If your are interested in the specifics check the small print on page 4 of the attached document.
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File Type: pdf TechNote_SeqCapEZ_TruSeq_15Apr2011.pdf (2.40 MB, 322 views)
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Old 05-10-2012, 05:19 PM   #14
ECO
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Thanks again. Asked my Nimblegen rep this same question and it would've been nice to see this year old document. Heh.
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Old 05-10-2012, 05:22 PM   #15
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I'm not sure why but NimbleGen removed this document from their website.
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Old 05-11-2012, 08:26 AM   #16
YW0712
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Thanks everyone for the info. I noticed that in the document above, the blocking oligos don't have a 3' terminator. Would that make any difference in the amplification or any other step?
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Old 05-11-2012, 11:51 AM   #17
DoubleA
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YW0712,

I have the same question. The blocking oligos I've been using do not have a 3' terminator. As noted previously, I observe a PCT_SELECTED_BASES of 55-65%. I often wonder if my PCT_SELECTED_BASES would be higher if my blocking oligos contained a 3' terminator. NimbleGen's most recent document on multiplex sequence capture (attached) recommends using blocking oligos with a 3' terminator.

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Old 06-11-2012, 03:20 PM   #18
ECO
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Update..."split" blocking oligos using 2'MO modifications brought the PCT_SELECTED_BASES to 52%.

I'm testing the 6xInosine and LNA-split configurations and will report back.
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Old 07-03-2012, 12:33 PM   #19
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I'm also wondering about 3' terminators. Has anyone directly compared using blocking oligos with and without terminators? Does carryover of the terminated oligos impair the subsequent PCR at all (i.e., by keeping the non-terminated primers from binding)?
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Old 07-06-2012, 02:09 PM   #20
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Hi guys,

If I can jump in... We used the v2 Roche exome kit for TruSeq libraries last fall and just did an overnight hyb for the training. We used the specific blocking oligo for Index 5 and back then they did not ask for terminators on the oligos...I think just a phosphorothioate bond.

Anyways, we got a nice enrichment with very little off-target material and decent enrichment by qPCR (>150x). And this was a non-standard workflow back then since Roche was not yet on board with TruSeq.

We recently used the Roche v3 kit with some samples with NuGen "TruSeq-style" adapters...we didn't have the correct blocking oligos so we just used the closest of the "demo" blocking oligos that Roche supplied. Unfortunately, we have a significant number of off-target (intron and intergenic) sequences in our final seq data. Close enough wasn't good enough, unfortunately. I need to do some target calcs...looks like you guys are using Picard tools for this.

Anyways, the point of this post is that we recently ordered the new Roche blocking oligos but just went with six N's where the index is...and we got the expensive terminator added on. I'm concerned that this won't work well, either but I'll post my results back here as I think this is the only thread of this type on here right now.

There has got to be a way to avoid buying a specific >$100 oligo every time we use a new index sequence.
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