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  • MiSeq for library QC

    Hello,

    Are you at a core facility that has a MiSeq? If so, are you using the MiSeq to quantify/QC libraries before processing on a HiSeq? If you do this, can you share how you have the workflow setup to make it cost effective? Currently, MiSeq runs are expensive (relative I know…), so how do you integrate this QC in to your workflow?

    Best,

    Anoja

  • #2
    Hi Anoja,
    Yes, I agree, while a quick MiSeq run would be an ideal check of the relative quality and quantities of libraries to be sequenced on a HiSeq, the cost of a MiSeq 50 cycle run is around $700 in reagents. Not so bad, if you could run every library that will be on the next flow cell or two. But with only 24 TruSeq indexes in the DNA and RNA kits, you will be lucky to fit 2-3 lanes worth of samples on the MiSeq.

    So, if we have some reason to worry about a library pool, we might burn a MiSeq run on that. But really what is needed is a very large number of bar codes so that 1-2 flow cells of sample could be meta-pooled and run on the MiSeq. The 96 index combinations available for Nextera are a step in the right direction. But as currently configured, Nextera is really only for DNA library construction.

    The thing is, in principle, you could whomp up a batch of 40 oligos using ECO's hybrid TruSeq/Nextera, ("TruSeqEra"?) strategy (maybe going to 10-12 nt indexes?) and anneal them row x col (for 40, I would do 24 x 16) == 384 dual-index combination adapters that should run fine on the MiSeq. Because of the their "TruSeq" nature, I think they should work fine on the HiSeq, but you might need to spike in some Nextera index sequencing oligos.

    --
    Phillip

    Comment


    • #3
      Hi Phillip,

      We have used BiooScientific Adapters (96 are available). Since you are at a core you understand the difficulty in batching samples/orders. Also, if the library failure rate is low and if pooling seems to be efficient, added steps might not be worthwhile.

      Do you have a MiSeq? Currently, what do you use it for?

      Best,

      Anoja

      Comment


      • #4
        BIOO work really well in our hands, and they have 96. My only (major) concern with relying on them is sustainability and continued quality from a small company. Hence my push to homemade.

        Philip if you are up for "helping" (with design and support...I can probably cover the funding), I'd love to order up a batch for people to test. I'd love to have a seqanswers-designed and -"validated" adapter set...

        Comment


        • #5
          Originally posted by ECO View Post
          BIOO work really well in our hands, and they have 96. My only (major) concern with relying on them is sustainability and continued quality from a small company. Hence my push to homemade.

          Philip if you are up for "helping" (with design and support...I can probably cover the funding), I'd love to order up a batch for people to test. I'd love to have a seqanswers-designed and -"validated" adapter set...
          Sure, count me in.

          --
          Phillip

          Comment


          • #6
            Originally posted by aperera View Post
            Hi Phillip,



            Do you have a MiSeq? Currently, what do you use it for?

            Best,

            Anoja
            We just got a couple of them. The most immediate use we are looking at is replacing our GS-FLX's functionality. We were mostly using the 454 for "Amplicon" work. That is, an investigator has some loci of interest that they would like sequenced over a number of samples at sufficient depth to discern all SNPs (or count them). But I think we would also push investigators to use it for smaller genomes -- small bacterial genomes or smaller just don't need a lane of HiSeq. So viral genomes, plasmids, BACs, etc. I would also love to see it further empty our Sanger sequencing pipeline.

            We're trying to focus on keeping turn-around under a week for the MiSeq. The HiSeq is just too much of a monster to allow this. And the problem is not just that the run times are long. It is the number of samples that need to be QC'ed, library constructed, and QC'ed again. The flow cell that finished yesterday had 72 samples on it. From 9 different labs. (Some of those labs had two or more projects on the run.)

            I am really hoping the 2500 chemistry/hardware works well out of the box. Because it solves so many problems being able to run what is, effectively, a quarter flow cell in about 1/8th or 1/10th the amount of time it takes to run a full flow cell.

            MiSeq has the potential to be a "gateway" next gen sequencer. That is a next gen instrument for the majority of researchers who have never attempted to undertake a next gen project.

            Towards that end it would be really nice if Illumina could deliver a MiSeq reagent set that produced the full length reads (2x150 now) at lower densities, same speed, but 20% of the cost. A 2x150 run that produced about 500 million bases of sequence (~1.5 million read pairs) for $200 in reagents. Or even less sequence. Whatever. Something to sit in the gap between doing a hand full of sequences on a Sanger sequencer, and being crushed by an avalanche of data from a HiSeq lane. (1 lane of 2x100 HiSeq produces as much sequence data as a top-end 96 capillary Sanger sequencer -- a 3730XL -- running for more than 50 years.)

            --
            Phillip

            Comment


            • #7
              Only way that is likely to happen is if there was a competitor/competing technology.

              Do you know of any other instrument/technology that could potentially achieve similar to what you are proposing?

              Originally posted by pmiguel View Post
              Towards that end it would be really nice if Illumina could deliver a MiSeq reagent set that produced the full length reads (2x150 now) at lower densities, same speed, but 20% of the cost. A 2x150 run that produced about 500 million bases of sequence (~1.5 million read pairs) for $200 in reagents. Or even less sequence.
              --
              Phillip

              Comment


              • #8
                Originally posted by GenoMax View Post
                Only way that is likely to happen is if there was a competitor/competing technology.

                Do you know of any other instrument/technology that could potentially achieve similar to what you are proposing?
                I had the idea that Ion Torrent had a chip that cost somewhere in that range. Don't they?

                Also, ABI seems to have completely abandoned innovating for the Sanger sequencing research community. Nevertheless they are sitting fat and sassy atop the bundles of cash they no doubt make from selling Big Dye and that crazy clean up kit. At some point, they become vulnerable, right?

                --
                Phillip

                Comment


                • #9
                  Sounds like there are no confirmed viable alternatives in the short term for your original wish.

                  Only way ABI may become vulnerable is when MiSeq/Ion Torrent gets FDA certification for clinical sequencing. But even then for single clinical samples ABI will still be the only choice.


                  Originally posted by pmiguel View Post
                  I had the idea that Ion Torrent had a chip that cost somewhere in that range. Don't they?

                  Also, ABI seems to have completely abandoned innovating for the Sanger sequencing research community. Nevertheless they are sitting fat and sassy atop the bundles of cash they no doubt make from selling Big Dye and that crazy clean up kit. At some point, they become vulnerable, right?

                  --
                  Phillip

                  Comment


                  • #10
                    Originally posted by GenoMax View Post
                    Sounds like there are no confirmed viable alternatives in the short term for your original wish.

                    Only way ABI may become vulnerable is when MiSeq/Ion Torrent gets FDA certification for clinical sequencing. But even then for single clinical samples ABI will still be the only choice.
                    GnuBIO & Qiagen (ex-Intelligent Biosystems) are the other near-term contenders for this market (perhaps LaserGen could also be considered). GnuBIO is strictly aiming at amplicon market initially, but could be competitive. If Qiagen's final instrument resembles the IBS box, then it could be a competitor for MiSeq.

                    Ion 314 chips I think are in the $99 range, but sample prep costs might push it out of the desired price range. There's also the indel issue -- for some assays this is a killer, for others it can be tolerated (e.g., if you know your variants are always missense, you can toss out the indels).

                    Comment

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