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  • #16
    Why not 2x301 on v2 kits?

    If you have a bunch of old kits you can just hack the 2x250bp v2 kits to run 2x301 bp.

    It works quite nice - we have been sequencing full V1-3 16S on MiSeq since April.

    You can see how we do here: http://midasfieldguide.org/docs/miseq_103.html

    If anyone wants to see some data or quality plots I can make a few datasets available.

    rgds
    Mads
    Last edited by MadsAlbertsen; 08-22-2013, 09:13 AM.

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    • #17
      That's a great hack Mads!

      A while ago I tried an asymmetric 301x201 run using a v2 500 cycle cartridge. Although the quality heat map looks OK, I wasn't impressed by the error rate after 250bp and its clear something bad is happening to the G and A base calls when you look at the %base plot. Lets hope they've got that sorted with the new v3 kits...



      Last edited by gwilkie; 08-22-2013, 10:17 AM. Reason: added pictures

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      • #18
        We do not see that dramatic error rate in the merged sequences.

        I've attached a quick plot of how the quality score usually looks after merging.

        rgds
        Mads
        Attached Files

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        • #19
          The %A bias is very suggestive you are running off the end of the fragment, so you would need longer insert libraries to benefit from the longer reads.

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          • #20
            Thanks Nick, I don't want to hijack this thread but maybe we should start another one about shearing for large insert libraries? I find it quite tricky shearing to larger sizes with a Covaris. Measuring library concentration by realtimePCR may also become problematic with larger insert sizes?

            This was a case where I had to use a 500 cycle cartridge that was getting near its use-by date for a run that I would normally have done as 2x150bp, so I decided to try the asymmetric long run as an experiment. I didn't think runoff from small fragments would be a major problem as most of the fragments were >250bp (see overloaded TapeStation trace below), however when I look back I can see that you are right - adapters were trimmed from about 20% of reads.




            Originally posted by nickloman View Post
            The %A bias is very suggestive you are running off the end of the fragment, so you would need longer insert libraries to benefit from the longer reads.

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            • #21
              Originally posted by epistatic View Post
              The PE-300 is better suited for amplicon and genome than exome and transcriptome. We'll have to fragment cDNA to get long enough fragments to do a PE-300 RNA-seq library, currently with TruSeq it is hard to get >150-300 bp fragments with 0-1 minutes fragmentation.
              No, it is fairly easy. There are plenty of long fragments when you use shorter fragmentation times, they are just mixed with short ones. If you lower your AmPure cuts, you can get rid of the the short ones and drive your amplicon lengths as high as you might need to.


              Oh, you might also have to increase the RT incubation duration back to 50 minutes from 15 minutes. The short RT incubation happened when they tried to shorten the protocol upon creation of the strand RNA kits.

              --
              Phillip

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              • #22
                Originally posted by gwilkie View Post
                Thanks Nick, I don't want to hijack this thread but maybe we should start another one about shearing for large insert libraries? I find it quite tricky shearing to larger sizes with a Covaris. Measuring library concentration by realtimePCR may also become problematic with larger insert sizes?

                This was a case where I had to use a 500 cycle cartridge that was getting near its use-by date for a run that I would normally have done as 2x150bp, so I decided to try the asymmetric long run as an experiment. I didn't think runoff from small fragments would be a major problem as most of the fragments were >250bp (see overloaded TapeStation trace below), however when I look back I can see that you are right - adapters were trimmed from about 20% of reads.
                This is not an issue that can easily be addressed at the shearing stage, instead you need a more effective size selection step. Or rather, a method that does a more effective job of eliminating the smaller fragments.

                I don't know why, but smaller amplicons have a massive advantage in clustering. So you have to completely eliminated them or they will cluster and displace the longer amplicons that you want.

                There are a couple of factors here:

                (1) Once you amp the library, some percentage of your short amplicons will anneal to the longer strands and become "shielded" from size selection. So do most of your size selection at earlier stages.

                You will still need to do a final size selection to remove adapter/primer dimers. But you should also minimize the number of cycles of amplification to avoid creating primer dimers.

                (2) Additional cycles of ampure cuts help. But you will also need to lower the ampure (PEG) % to remove all but the larger amplicons.

                (3) If you decided to cut of an agarose gel, do no heat the gel slice! If you use some sort of chaotrope to melt the agarose, just use more of it and the slice will melt at room temp. Yields are much higher this way.

                (4) When you assay your library prior to clustering, just realize that the left side of your peak is what you end up sequencing. Again, I don't know why, but results always seem to be as if all the amplicons queued by size prior to moving to the flow cell surface, with all the longest amplicons never reaching the surface. I can't think of a mechanism that would cause this to happen, but empirically, that is what I see.

                --
                Phillip

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                • #23
                  Originally posted by pmiguel View Post

                  I don't know why, but smaller amplicons have a massive advantage in clustering. So you have to completely eliminated them or they will cluster and displace the longer amplicons that you want.
                  From my understanding talking with my FSA about this, part of the reason that smaller fragments cluster better is that they can more quickly and efficiently build compact clusters compared to large fragments. He seemed to imply that this was due to shorter fragments being more efficient at forming the bridges, so over the course of the entire cluster generation phase, the shorter fragments out compete the larger ones for access to the anchoring oligos and thus those clusters become the predominant ones on the flow cell.

                  I would also suspect that shorter fragments have an advantage simply because there's less non-adapter sequence to have to deal with, so the anchoring oligos and the adapter sequences are able to anneal together more quickly.

                  Lastly, as Illumina says, fragments larger than 1Kb can supposedly bridge both surfaces of the flowcell, which would mean they wouldn't form clusters at all. I can't say if I really believe that, because a 1Kb stretch of DNA is pretty small and it seems like the two surfaces are further apart than that, but it could be occurring...

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                  • #24
                    sorry for such strange question, but what is the colour of incorporation mix in Miseq kits v.3?
                    I got our first v.3 kit today and I'm very much surprised to see that incorporation mix is dark blue, not rose-violet, as in v.1 and v.2 kits. Is this normal?

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                    • #25
                      Hi Mlog,

                      MiSeq V3 kits have updated chemistry. One of the most obvious changes is that the incorporation mix is now blue instead of purple.

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                      • #26
                        This kit seems perfect to sequence a 300Mb fugu genome.

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                        • #27
                          MadsAlbertsen, would you mind reposting a link to the hack protocol? Your previous link has now removed the hack details as "the extra reagent in the v3 kit makes it unnecessary"... however recently it seems there is an issue with amplicon sequencing and R2 quality on the MiSeq using v3 chemistry and we cannot follow Illumina's advice of shortening our amplicon to <500bp (which they are recommending as the v2 chemistry doesn't seem as affected). I was therefore wondering why they don't just supply v2 kits with 2x300bp capacity, and figured I might give your hack a try.

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