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  • #46
    Some references>
    Okou et al. Nat Methods. 2007 Nov;4(11):907-9
    Excelent method description in the supplement

    Hodges et al. Nat Genet. 2007 Dec;39(12):1522-7.
    Describe the sequence capture followed by Illumina Genome analyzer

    The custom made sequence capture arrays from Nimblegen now come with full protocol for the sample prep, should work for Illumina if you just use the right oligos.

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    • #47
      first experience with nimblegen seq cap on GAII - ?problem


      Edited 10 SEPT 2008: I wish to withdraw this post - see post 48 below:


      Hi,
      We've just run our first nimblegen seq cap service (i.e. they did the probe design/hyb/elute/pcr amp steps) sample on a solexa flowcell. We confirmed the returned sample looked OK on bionanalyser.
      No problems with solexa library prep.
      Flowcell worked well (GAII) for other libraries.
      After removing the 454 adapters, almost no reads map to the regions we asked for on the chip (custom design 1.5Mb). However the reads map very well to the human genome (maq alignment to whole genome, >90% map, error rate 1.0%). But to a few distinct regions which look like they might be repeat regions.

      We have some other samples to run, and will repeat the library prep, but am a bit concerned by this. Wondered if anyone else has had any difficulties?

      david
      Last edited by dvh; 09-10-2008, 12:46 PM.

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      • #48
        first experience with nimblegen seq cap on GAII

        I wish to withdraw the post (#47) above.

        There was a sample mix up on the flowcell. Lane 1 was actually lane 8 etc. Found this out from the PhiX control, the fact we had mixed sample types, and careful inspection of the data. Worth pointing out to the list that it is VERY easy to flip the 8-well tube of samples (or perhaps flowcell) in the cluster station.

        To apologise for the earlier post, here are some results from the first lane we've looked at. Single end solexa reads. We have only very crudely dealt with the (unwanted) adapter sequence used by nimblegen for PCR amplification at present, much more optimisation is possible which will improve both number of reads and quality of reads aligning. Using maq map -n 3 to the whole genome, 1.3m reads map with MQ 70-99 i.e. uniquely with high confidence. Of these 85% map to the regions we selected for the seq cap chip. So it works. Somewhat variable coverage, I'll try and post some stats once we've done some more analysis.



        david

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        • #49
          Hi all!

          My first time here so be gentle
          My question is: have you any idea whether the sequencing results will allow the user to distinguish strand orientation... This because I am interested in transcriptome analysis and the strand info is crutial... Let me know please, I'm drowning in papers...
          Cheers to all and thanks to the threading you all are introducing me to a brand new world!
          ciao!

          Comment


          • #50
            Not sure if I understood you right, but Yes. The alignment algorithm will tell you if the sequence aligned to forward/reverse strand of the reference sequence you provide..
            --
            bioinfosm

            Comment


            • #51
              Originally posted by poliveira View Post
              Hi all!

              My first time here so be gentle
              My question is: have you any idea whether the sequencing results will allow the user to distinguish strand orientation... This because I am interested in transcriptome analysis and the strand info is crutial... Let me know please, I'm drowning in papers...
              Cheers to all and thanks to the threading you all are introducing me to a brand new world!
              ciao!
              You will only be able to tell the direction / strand of the sequence you read, not the transcript. If you have polymorphisms in your exons you might see some strand specificity though. But for transcriptiome sequencing you do not need the enrichment arrays I guess.

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              • #52
                Hello,

                HD2 is available from Nimblegen !

                Anyone have more information about that ?

                Comment


                • #53
                  Nimblegen are hosting a webinar very soon. See here.

                  Comment


                  • #54
                    imp[rovements for the library prep in this paper:
                    Nat Methods. 2008 Dec;5(12):1005-10.

                    A large genome center's improvements to the Illumina sequencing system.

                    Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H,
                    Turner DJ.

                    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
                    Cambridgeshire, CB10 1SA, UK.

                    Comment


                    • #55
                      Hi all, do you know an alternative method to perform a microarray experiment using 454 Junior? (no Nimblegen)
                      Thanks

                      Comment


                      • #56
                        An urgent help needed regarding sequence caputure

                        Hi.....
                        I want some information regarding nimbulgen sequence capture of mouse genome.I want to sequence4.5 Mbp region on x chromosome of few mouse samples.can anybody share there experience with mouse samples.All kind of recommendations are welcome.Please send me some references or protocols or articles on the same.Please help.
                        Best regards,
                        Tanmoy

                        Comment


                        • #57
                          Hi
                          We have done Nimblegen sequence capture on dogs, horses, cows, mouse, humans, etc. All works fine. Just using the standard protocol from Nimblegen+Illumina should work fine, or use your own library preparation variants.

                          Comment


                          • #58
                            Hi sigusn....
                            Thank you for your reply....can u suggest me some research papers.
                            rgds,
                            T

                            Comment


                            • #59
                              We don't have anything published on Mouse yet. But here are some papers:
                              This one tests Nimblegen capture
                              Although oligonucleotide arrays are quick and easy to develop, some problematic regions may evade capture, necessitating sequential redesigning for complete optimization. Neither sequencing technology was able to detect every variant identified by Sanger sequencing because of well-known drawbacks of …

                              Using Nimblegen capture on Horses
                              We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromo …

                              On cattle
                              PMID: 20865119
                              Nimblgen has a collection of papers, user guides and protocols

                              I hope that helps something
                              regards,
                              Snaevar

                              Comment


                              • #60
                                thank you very much for the help.
                                regards,
                                Tanmoy

                                Comment

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