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  • sigusn
    replied
    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.

    Leave a comment:


  • sci_guy
    replied
    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.

    Leave a comment:


  • sigusn
    replied
    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. I´m 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.

    Leave a comment:


  • seqer
    replied
    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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  • imera
    replied
    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.

    Leave a comment:


  • ivoire
    replied
    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.

    Leave a comment:


  • Rosalind_F
    replied
    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

    Leave a comment:


  • ivoire
    replied
    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

    Leave a comment:


  • Rosalind_F
    replied
    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.

    Leave a comment:


  • sci_guy
    replied
    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.

    Leave a comment:


  • seqer
    replied
    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?

    Leave a comment:


  • ECO
    replied
    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.

    Leave a comment:


  • sci_guy
    replied
    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

    Leave a comment:


  • seqer
    replied
    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!

    Leave a comment:


  • sci_guy
    replied
    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.

    Leave a comment:

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