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  • #31
    There a small mistake on the picture. If we saw sequences of adaptors, primers and oligonucleotides
    (Accurate whole human genome sequencing using reversible terminator chemistry.
    David R. Bentley, Shankar Balasubramanian etc.
    Nature, Vol 456 | 6 November 2008 | doi:10.1038/nature07517)
    --- we can see that fragment links with oligonucleotide on the surface of flow cell not with adaptor but with additional sequences in primers, added by PCR.

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    • #32
      Originally posted by anju View Post
      simple comparison
      Pretty out of date, too. Anything with the latest HiSeq, Solid 4, and "1000-base" 454?

      I'd love to see some info on useable read length, too.

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      • #33
        hello all I am very,very, very new to NGS. trying to learn as much as I can

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        • #34
          Bridge Amplification

          Hi All,
          I'm new to this whole NGS business. I will be using an Illumina GAII. In a recent article from Bio Techniques, I read that longer (about 600bp) fragments can increase the uniformity of coverage across a targeted region. In the paper, amplicons were fragmented to 200bp prior to adaptor ligation and subsequence sequencing. Does anyone know why this would increase uniformity? Also, does anyone know if it is possible to successfully generate clusters during bridge amplification with fragments larger than 200-300 bp? And if so, how large?

          THANKS!!

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          • #35
            Is this material applicable to Illumina's systems today, or does it need updating?

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            • #36
              Originally posted by rzhuo View Post
              Hi there,

              I have a question about the software I can use to analyze the Illumina sequencing data. I'm doing targeted resequencing of long PCR fragments to find some EMS mutations. I'm waiting for my run to finish right now and meanwhile I'm looking for a suitable software. Any suggestion would be of great help to me. Thank you.
              Probably north of 95% is still perfectly applicable. The only major changes are to the chemistry robustness which increases yield and readlengths. I don't personally run any form of ILMN machine so I welcome correction from an Illumaniac*.


              *New name for the Illumina forum!

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              • #37
                Will another forum be renamed to "On SOLiD Ground?"

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                • #38
                  Thanks for the useful information

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                  • #39
                    Hello, I´m new in NGS. I want to sequence target regions using human genomic DNA. My purpose is to sequence de coding regions of 400 genes (around 1Mb). Which is the best approach?
                    In addition could you recommend me what software can be easily used to analyze data from exome assays.
                    Thank you.

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                    • #40
                      Wrong thread. You'll get more eyes & brains on your question if you post in a more appropriate area (and say how many samples you are working with & what sort of species this is for)

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                      • #41
                        question... so paired end reads... are they from the same strand or the opposite??

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                        • #42
                          Nobody ever answered the original question, so here goes:

                          The fluorescent bases are 3'-O-azido dNTPs with fluorophores linked to the bases. The azide group on the 3' O blocks addition of another nucleotide, and you can use a phosphine (TCEP) to chemically cleave the azide from the 3' O and allow the next nucleotide to be added. I believe TCEP also cleaves the fluorophore from the base.

                          It's very clever, but unfortunately 3'-O-azido dNTPs are not available commercially. They would be fun to play with.

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                          • #43
                            pair-end sequence poplar transcriptome

                            Hi there,

                            I wanna do mRNA-seq on poplar, I review a bunch of literature without finding a single paper using pair-end sequencing, most of them use 454 sequencing. Does that mean pair-end sequencing does not work on poplar? Any suggestion?

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                            • #44
                              Originally posted by li zhang View Post
                              Hi there,

                              I wanna do mRNA-seq on poplar, I review a bunch of literature without finding a single paper using pair-end sequencing, most of them use 454 sequencing. Does that mean pair-end sequencing does not work on poplar? Any suggestion?
                              What about these articles? Whole genome, transcriptome, exome?
                              What genome size? How many groups work on poplar sequencing?
                              Best results for de novo assembly from 454, of course. But for resequencing why not use pair-end read?

                              Comment


                              • #45
                                hi all,
                                Does anyone knows about illumina data downloadble from any published papers?
                                many thanks

                                Comment

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