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  • #16
    Originally posted by colicoli View Post
    My guess for the reason for the long hybs to Nimblegen arrays is that at 42 degrees, it takes a LONG time for the hybridization reaction to approach equilibrium. While short oligos can hyb quickly in solution, the oligo probes on the array are fixed and immobile, so the reaction relies on diffusion of the target genomic DNA fragments. These target DNAs are at least several hundred nt, and it will take each one a lot longer than overnight to find its complement on the array surface, esp. if the concentration is limiting (note the large sample requirement in these protocols). It would be much faster if the targets were short, like microRNAs. The combination of large sample+slow hyb times for the array based methods may give an edge to solution-based capture methods if the bugs get worked out (the solution capture probes could find their targets more quickly than fixed capture probes).
    I would bet that the 42 degree hyb temperature could be determined not by biochemistry, but by engineering. For Nimblegen probe lengths, the hyb could more stringent (and diffusion is faster) at 65 degrees, but its harder to contain a small volume of liquid at a higher temperature for a long time.
    Very good points colicoli! I still dont quite understand hyb at 42C. It seems that the specificity looks okay (at least in Albert et al). Intuitively, 42C hyb would generate a lot of nonspecific binding.

    Also, does anyone have an idea how much amount of oligos per feature (spot) on Nimblegen array? It seems that it is extremely low so that they need 3 day hyb time and 20ug of gDNA to drive the kinetics.

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    • #17
      Originally posted by sci_guy View Post
      I've been in contact with Roche and I am lead to believe that a Nimblegen sequence capture service will be available around March, not long after human custom HD2 chips hit the market.

      I have some open-ended questions regarding this "new and improved" formula

      The HD2 array in order to go from 385K probes to 2.1M takes up much more real estate on the slide but also incorporates a feature shrink, from 16 to 13 micron. Presumably, there are less total oligos in this smaller 13 micron region. Will the feature shrink and increased numbers of probes make HD2 "noisier" than the 385K chip? Will we still see reasonable specificity?
      Why have Hodges et al. (2007) used a custom sequencing primer approach to their downstream Solexa sequencing? Will the 1G instrument handle a longer primer OK? Why do they not include the Solexa adaptor as an adaptor in the Nimblegen protocol?
      I ask these questions as I would like to capture 500 bp fragments on the Nimblegen chip and then alter the protocol to reduce the fragment sizes down before a SOLiD or Solexa/Illumina run.

      I'm in the process of emailing Solexa and the corresponding authors of these papers to get some answers. If you can inform me on any of this, please post!
      What are the answers?

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      • #18
        Response to sci_guy and horigen

        Originally posted by horigen View Post
        What are the answers?
        I'm not sure if you ever received the answers from Solexa or the authors you contacted, but I'm happy to answer. In the Hodges et al. paper, the DNA was prepared in two different formats, the Roche Nimblegen way (fragment sizes 500-600 or more bp) - which is suitable for 454 (conveniently since both 454 and Nimblegen are owned by Roche). Also, for these experiments common linkers were chosen based on previous experience (the sequence ligates well and amplifies well) and the fact that they do not add a lot more sequence onto the already large fragments. Having these linkers requires that additional adaptation be performed for Illumina sequencing. Therefore, the custom sequencing primer had to include the first set of adaptors, as they are the most internal and adjacent to the insert to be sequenced.

        In the same paper, an alternative DNA preparation was also reported. It turned out to be much more suitable for Solexa, and bypassed the extra ligation step. Basically, the libraries were prepared according to the Solexa kit protocol prior to hybridization and the fragment sizes were much shorter (150-300), a much more suitable size for short read platforms. This resulted in 90% base pair coverage and a pretty high sequencing depth for such a large target size (6 Mb of one exon chip).

        Most importantly, it is imperative to have the DNA fragments ligated to common oligo adaptors before hybridization. The reason for this is that thermal elution of the slides at 95 degrees, may result in oligo detachment. Therefore, having the DNA ligated with common oligos allows enrichment of the genomic DNA and avoids sequencing of oligos and non-specific material.

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        • #19
          Reseq: Many thanks for the info!

          Horigen: No one got back to me. But I have heard anecdotally that Nimblegen is on track to offer 385,000 probe chip enrichment as a service around the end of March. There are some delays with the HD2 chip. Agilent will be releasing liquid phase sequence capture product later this year.

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          • #20
            It seems to me that shearing the genomic DNA is quite critical. If one can reliably shear DNA to ~100bp or 200bp, the genomic DNA will move faster (thus increase hybridization efficiency), and the library construction for sequence whole exon is easier (one may simply sequence ends of a fragment).

            Anybody has a robust protocol to shear human genomic DNA to 100-200bp? Thanks!

            Comment


            • #21
              Ultrasonicators

              Originally posted by seqer View Post
              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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              • #22
                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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                • #23
                  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?

                  Comment


                  • #24
                    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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                    • #25
                      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.

                      Comment


                      • #26
                        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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                        • #27
                          Originally posted by ivoire View Post
                          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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                          • #28
                            Originally posted by Rosalind_F View Post
                            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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                            • #29
                              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.

                              Comment


                              • #30
                                Originally posted by imera View Post
                                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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