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Old 02-28-2008, 08:27 PM   #21
sci_guy
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Anybody has a robust protocol to shear human genomic DNA to 100-200bp? Thanks!
Yes. As I've found out either you, or your Next-gen service provider, needs a pretty funky new sonicator. It seems nebulisation won't cut it (bad pun ) for 100 - 200 bp libraries. See these pieces of hardware:
Covaris
Bioruptor
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Old 02-28-2008, 08:36 PM   #22
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Every user I've talked to has, or wants, the Covaris.

They have partnered with ABI...so it can't be a bad instrument. http://www.covarisinc.com/press_12.htm

I still feel that a well timed DNase reaction would be nearly as good, and shouldn't require filling in. But I guess it would be finicky.
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Old 02-28-2008, 08:58 PM   #23
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Thanks sci_guy and ECO! This thing is damn expensive... For some reason, people believe physical shearing / sonication is less biased than any enzymatic reaction. It might be worth trying DNase. Any known bias?
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Old 02-28-2008, 09:06 PM   #24
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If your favourite DNase works processively then I imagine will you see a big old smear on the gel. If the enzyme is non-processive and just creates dsDNA breaks then DNA size will be more tightly distributed with size a function of temp, salt and time.

No idea about bias.
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Old 03-20-2008, 08:45 PM   #25
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Has anyone tried to release oligos from an array, and if so do you have a good protocol? As I understand it, Church's group used a NH4OH, which simply requires a base-labile linker. And (forgive my many questions) would Nimblegen arrays be amenable to the same process?

Thanks in advance.
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Old 03-21-2008, 01:47 PM   #26
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Quote:
Originally Posted by Rosalind_F View Post
Has anyone tried to release oligos from an array, and if so do you have a good protocol? As I understand it, Church's group used a NH4OH, which simply requires a base-labile linker. And (forgive my many questions) would Nimblegen arrays be amenable to the same process?

Thanks in advance.
Nimblegen arrays are reusable. the capture arrays do not need to be stripped to be reuse if you follow their protocol to elute the fragments that are captured. As for their resequencing array, they sell a stripping kit that is used to strip the hybridized fragment off the chips. I have however successfully used their stripping kit on both the capture and the resequencing array (2nd use) without any significant effect on the results when compared to the first used array. You can order the stripping kit from them. it is called "Nimblegen Array reuse kit". good luck
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Old 03-26-2008, 11:37 AM   #27
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Nimblegen arrays are reusable. the capture arrays do not need to be stripped to be reuse if you follow their protocol to elute the fragments that are captured. As for their resequencing array, they sell a stripping kit that is used to strip the hybridized fragment off the chips. I have however successfully used their stripping kit on both the capture and the resequencing array (2nd use) without any significant effect on the results when compared to the first used array. You can order the stripping kit from them. it is called "Nimblegen Array reuse kit". good luck
I appreciate the information. Thanks.

Do you know if you can actually strip the oligos from the chip, not just the hybridized fragments?


R
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Old 03-26-2008, 05:58 PM   #28
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Quote:
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I appreciate the information. Thanks.

Do you know if you can actually strip the oligos from the chip, not just the hybridized fragments?


R
Nimblegen claim that their stripping kit preserve the probes. From my experiments, this seems to be true. The first and second use(I tested both the capture and the resequencing array) both provide basecalling results from 98 to 99%. I have gotten result where the second use gave higher basecall results than the first one, suggesting that. the performance of the third use is in lower but still over 90 % basecall. the resequencing array is more robuts than the capture array as it is subject to less heat.
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Old 06-19-2008, 12:48 AM   #29
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Regarding costs of Sequence Capture, I just got a call from NimbleGen offering full service Sequence Capture of 5 samples for $5000. However it only applies to human and mouse samples. The rest of us working with non-model samples still have to do all the footwork ourselves...Maybe in the future they will be a bit more flexible.
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Old 06-19-2008, 11:09 AM   #30
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Regarding costs of Sequence Capture, I just got a call from NimbleGen offering full service Sequence Capture of 5 samples for $5000. However it only applies to human and mouse samples. The rest of us working with non-model samples still have to do all the footwork ourselves...Maybe in the future they will be a bit more flexible.
$5000 total or each? It is hard to imagine they are doing $1000 each - the array would cost at least $600.
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Old 08-06-2008, 01:36 AM   #31
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Hi
I have just tested the Nimblegen Sequence capture for Solexa sequencing and it works fine if you have good quality 20ug input DNA. WGA samples worked but with more variation in the sequence coverage. Im not working with human/mouse but they have no problem of making custom arrays for different species. The only issue I find is that some repeat regions have not been removed and are sequenced >1000x compared to non-repeat regions with >50x coverage. This could also be issue with the alignment software (MAQ).
I used the Bioruptor for DNA sonication, had some problems to get uniform fragmentation of the DNA. Seems to depend on the quality of the DNA. Any questions or suggestions, feel free to contact me.
SS.
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Old 08-06-2008, 01:49 AM   #32
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Hi Siqusn,

So you don't see a large decrease in the number of reads per flow cell lane like that reported by Hodges et al.? I would be very interested in more detail!

We have ordered the Nimblegen seq-cap as a service and are weighing up Illumina GA-II and Roche 454 for sequencing. Since 454 "Titanium" has been delayed until November we are favouring the Illumina platform. The big question for us is if the chemistry changes and cluster detection changes since GA-I have allowed handling of longer Nimblegen seq-cap amplicons effectively. Otherwise we may go down a messy concatamerisation approach.
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Old 08-06-2008, 01:54 AM   #33
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I sonicated the DNA to 100-300 bp, followd the Illumina protocol for adding adapters, then seq-cap with Nimblegen arrays, PCR and Sequenced on Illumina GA-1. It worked out all right. I have not compared the exact number of reads to Hodges et al, but I got 4 million per lane.
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Old 08-06-2008, 06:46 PM   #34
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Hodges et al. reported that following something approximating your method they could get good Illumina GA-1 reads but suffered from poor sequence capture, whereas 500-mers could be captured OK but suffered from poor numbers of reads. Do you note sequence capture was less than optimal with your 100 - 300mer library?
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Old 08-07-2008, 02:02 AM   #35
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Hi
Hodges et al write “For optimal sequence generation on the Illumina platform, fragments of less than 250 bp in length are desirable. This was below the size range initially optimized for capture. ” But hen they could optimize the size range, they don’t mention that?
Im not sure how Hodges et al define “specificity of the capture” they write. “…found a reproducible threefold decrease in the specificity of the capture with shorter fragments (average size of 100–200 bp )(Table 2) However, this was more than compensated for by the increased sequencing efficiency and the broader distribution of fragment ends ”
What I can read from the tables is that in Table 1 they get from 100 K to 2 million reads per array and about 30-55% of the reads map to the selected exons. In table 2 they get 1,85-2,3 million reads and around 30% of the reads in selected regions.
In my data, I get 4-5 million reads per sample (lane) and the fraction of sequence reads that align to the captured region varies between 32-56% (same array different DNA samples).
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Old 08-07-2008, 02:26 AM   #36
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Excellent! Thank you Siqusn. Those numbers are informative for us. One last question, are you capturing a contiguous region (less Windowmasker repeatmasked regions)?
We are capturing a contiguous 5 Mb region. Nimblegen suggest they have about 70 - 80% specificity with standard ~500-mer library preparation and tiling over a large area.
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Old 08-07-2008, 02:32 AM   #37
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Yes Im capturing 6 separate contiguous regions total 1.25 Mb. If you are doing the seq-cap as a service at Nimblegen, will you have the option of using shorter fragments with Illumina adapters, or do you have to go with their standard protocol of 500 bp with 454 adapters?
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Old 08-07-2008, 02:44 AM   #38
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We are bound by their standard protocol I think. The service specifies delivery of 21 ug of DNA and they do the library prep and QC. The concatamerisation/shear/ligate Illumina adaptor method post-capture seems very messy from an in silico point-of-view if you consider that a high proportion of the reads will contain adaptor sequence starting at an essentially random location within a read; particularly so for mutation discovery.
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Old 08-11-2008, 02:42 PM   #39
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Hi Sci Guy and Sigusn,
I am also trying to do a seq. capture/GAII approach similiar to the Nimblegen arrays using a cdna array. Sounds like I'm doing something similiar to Sigusn, as far as using 100-300bp sonicated dna and following Illumina's prep for end repair/adaptor ligation. Next step is to hybridize to a cdna array, elute and then continue with Illumina's protocol. I wondering how you did the elution step. Thanks for your help.
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Old 08-12-2008, 12:49 AM   #40
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Hi Lambroso
The elution step is well described in the Okou et al. paper. (Nature Methods 2007). However i did not have any equipment from Nimblegen for this. So I used home made system, cutout aluminum block, for holding microarray slides and silicon rubber (Elastosil RT 601 A/B, Wacker-Chemie GmbH, Munich, Germany) that I cut out to fit the slide to minimize the area that I pipetted water on. Then placed this on a heat block and hoped it would stay at 95C wile I pipetted 400 ul H20 on the slides three times. Of course 1/4 of the water evaporated before I could remove it from the slide, but that should not be a problem. I just received my next batch of slides from Nimblegen, and now they come with a special unit for the elution step. How do you plan to perform the elution?
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