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  • How does insert size affect result?

    There are two PCR-free library prep kits from Illumina. One is 350bp insert. The other is 550bp.

    Based on my limited understanding, I think longer insert size does a better job for phasing. But longer insert size might have lower sequencing yield for low quality input. Does that sound about right?

    What else is different for these two libraries?

    Thanks a lot in advance

  • #2
    Originally posted by ymc View Post
    There are two PCR-free library prep kits from Illumina. One is 350bp insert. The other is 550bp.

    Based on my limited understanding, I think longer insert size does a better job for phasing. But longer insert size might have lower sequencing yield for low quality input. Does that sound about right?

    What else is different for these two libraries?

    Thanks a lot in advance
    Not sure what you mean by "lower sequencing yield". Either insert size works fine on Illumina machines. We nearly always use the 550bp insert size -- it is pretty much better for everything.

    --
    Phillip

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    • #3
      I agree, a longer insert is generally better. I don't suggest using aiming for short inserts except when you specifically want the reads to overlap for merging.

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      • #4
        I've used the 550bp insert protocol a few times with great results. Even though the resulting library concentration has never been close to 4nM I've still been able to cluster at close to max capacity on the Miseq v3 flow cell.

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        • #5
          Originally posted by pmiguel View Post
          Not sure what you mean by "lower sequencing yield". Either insert size works fine on Illumina machines. We nearly always use the 550bp insert size -- it is pretty much better for everything.

          --
          Phillip
          Thanks for telling me my mistakened thought. I thought bigger fragments are generally fewer after shearing in theory. But I suppose 350bp and 550bp doesn't make much difference in practice.

          Comment


          • #6
            Originally posted by Brian Bushnell View Post
            I agree, a longer insert is generally better. I don't suggest using aiming for short inserts except when you specifically want the reads to overlap for merging.
            To overlap a 350bp, you need a 2x250 or more run. But mostly people run 2x100. Does that mean there is no point of using 350bp? Is the 350bp kit cheaper than the 550bp kit?

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            • #7
              You can merge 350bp inserts via overlap with a 2x200 run. But you don't really need to merge in order to do phasing; with the appropriate software, unmerged paired reads should do just as well. That said, I'm not sure what the appropriate software is.

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              • #8
                Generally you do 2x100 because that is read length supported by Illumina for a highoutput HiSeq2000/2500 run. For many machines that gives you the lowest price per base.

                For the most part cost/base from a HiSeq beats what you can obtain from a MiSeq by a wide margin. So those long MiSeq reads have to be quite valuable to spend the extra $/base on them. I've often seen it stated that 2x250 or 2x300 base reads are better for de novo sequencing. But I've not seen much of an advantage for using them over 2x100 in practice. At least I rarely see much improvement in a de novo genomic assembly of 2x100 reads from adding a 2x250 or 2x300 MiSeq run. Adding reads with longer spanning distances, such as a mate pair library seems to be much more valuable in that regard.

                This seemed to be true even back in the Sanger sequencing days with BAC-sublibraries. I remember sometimes someone would overshoot on the insert size of their plasmid sublibraries so we ended up with 9-12kb inserts in high copy number vectors. Plasmid yields would suck because the host strain was choking long plant DNA inserts (barley or wheat) in a high copy number vector. But those paired reads would assemble these gnarly BAC sequences way better than shorter sub-clones.

                Generally amplicon runs are the main place where spending extra on read length can be worth it. And, of course, if cost/base decreases with read length -- as it does with v4 highoutput chemistry, rapid chemistry, or I would imagine, HiSeq3000/4000 runs, then that's win-win for many applications.

                --
                Phillip

                Comment


                • #9
                  If I am doing PCR-free metagenomics stuff that might have virus in the sample, should I use 350 or 550? I heard that fragments of virus DNA is often short, so maybe 350 is preferred?

                  Comment

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