[YW0712]

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?

[DoubleA]

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.

DoubleA

[ECO]

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.

[mlcarpen]

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)?

[GeGnome]

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.

[SeqTruth]

Hi All,

I have also been curious about whether it's possible to use TruSeq or other 3rd party lib prep methods in conjunction with SureSelect. I understand the importance of using the appropriate blocking oligos for the adapters used, but I was wondering about an even simpler issue: is it possible to order the SureSelect capture kits without ordering a matching lib prep kit? I know they have separate part numbers, but their website gives the impression that they must be ordered together.

[DoubleA]

Hi SeqTruth,

We have purchased both Agilent SureSelect and NimbleGen bait libraries without ordering a matching library prep kit. We use a "home-brew" protocol to produce libraries with 64 barcodes. For the SureSelect hybridizations, we use their bocking oligo mix that comes with the kit. For the NimbleGen bait libraries, we purchased HPLC purified blocking oligos with deoxyinosines at the barcode positions and a phosphorothioate bond at the end. We get 55-70% reads on-target (Picard) using baits from either company.

DoubleA

[ECO]

I have also been curious about whether it's possible to use TruSeq or other 3rd party lib prep methods in conjunction with SureSelect. I understand the importance of using the appropriate blocking oligos for the adapters used, but I was wondering about an even simpler issue: is it possible to order the SureSelect capture kits without ordering a matching lib prep kit? I know they have separate part numbers, but their website gives the impression that they must be ordered together.

I have been told by my rep that they now sell the baits separately...without the library kit. The wrinkle here (and a continued annoyance) is that there is no way to get the hyb reagents as well...without resorting to a custom order. It is possible however to get JUST the library kits if needed.

[gevro]

Hi ECO,
Have you had a chance to see how the 6-I or other modifications compare? It is quite an expense to order the full blocking oligos, so if the 6-I works decently we might do that. We saw also ~45% with split oligos, which is not quite good enough for us.

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.

[BuckeyeSeq]

Hey ECO,

Our lab is looking to complete a truseqDNA library prep combine with a targeted exome capture using Agilent's hybridization kit. We have blocking oligos (from roche's blocking oligo kit) for each truseq index adapter. We are curious to the exact make-up of your hybridization reaction. I am assuming the hybridization buffer mix is the same and that you have altered the Sureselect block mix.

You stated that you do not add sureselect block #3, but do you add suresect block #1 and #2? How much of the specific truseq blocking oligos do you add?

I appreciate the help!

[sverma]

Hi All,

I saw there was a lot of discussion on this thread back in 2012, but, then it has no posts since then. I am doing sureselect xT capture and using their own library prep kit and blockers I am getting only 40% on-target capture. I am also looking in to some home brew versions of the adapters and blockers. If anyone who has a protocol that works with home-brewed library prep method and sureselect capture can they share that on the thread. Also, ECO I saw that you were trying 6 Inosine modification on index adapter blocker in 2012. Did that work? Can you please share the results on thread.

Thanks,
Suman

[zhuangran]

Hi, ECO. I am wondering how to determine the concentration to use of the newly designed
"Block #3". What I am now using is Agilent SureSelect XT Target Enrichment System (not pooling version), and it required in total 750ng DNA as input for hybridization and 0.6ul Block #3. As I wanted to adapt this protocol , how much adapted "Block #3" should I add? Will 50uM equal amount mixture be sufficient?