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  • Multiplexing beyond 96

    I've been looking at doing some shotgun metagenomic sequencing on the HiSeq 2500 (or 3000/4000). It seems that with both Nextera DNA and Truseq Nano (or PCR-free) Illumina only has indexes that allow multiplexing up to 96 samples. I would, ideally, like to multiplex far beyond this, to 200 samples or more.

    Does anyone know of a third party company that makes additional indexes for Truseq or Nextera DNA that will allow me to multiplex higher? Or has anyone made their own indexes?

  • #2
    If you make your own barcodes, be sure to validate them somehow (qPCR? actual sequencing?) because not all sequences work equally well. Usually it's safer to copy someone else's validated barcodes.

    Fluidigm has a set of 384, which use 10 nt single-indexing. I'm not sure you can buy the oligos, but at least you can synthesize them.

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    • #3
      Originally posted by cheezemeister View Post
      I've been looking at doing some shotgun metagenomic sequencing on the HiSeq 2500 (or 3000/4000). It seems that with both Nextera DNA and Truseq Nano (or PCR-free) Illumina only has indexes that allow multiplexing up to 96 samples. I would, ideally, like to multiplex far beyond this, to 200 samples or more.

      Does anyone know of a third party company that makes additional indexes for Truseq or Nextera DNA that will allow me to multiplex higher? Or has anyone made their own indexes?
      You can get Nextera XT index primers from Illumina for 384 dual indexing. It comes in 4 sets of 96.

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      • #4
        Originally posted by nucacidhunter View Post
        You can get Nextera XT index primers from Illumina for 384 dual indexing. It comes in 4 sets of 96.
        Illumina says Nextera XT indexes cannot be used with Nextera DNA, only with Nextera XT.

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        • #5
          Originally posted by jwfoley View Post
          Fluidigm has a set of 384, which use 10 nt single-indexing. I'm not sure you can buy the oligos, but at least you can synthesize them.
          I was hoping to copy someone else's validated barcodes, as you suggest, as validating a barcode set that large could be very time consuming and expensive. If a solution is commercially available, I'm certainly open to buying it.

          Fluidigm provides the index sequences in that document. Would I just take the Illumina TruSeq adapter sequences and replace their index sequence with Fluidigm's?

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          • #6
            Originally posted by cheezemeister View Post
            Illumina says Nextera XT indexes cannot be used with Nextera DNA, only with Nextera XT.
            I have used them across both kits and data looked fine. You may want to try on a small number of samples. There is no barcoded primer out there that has been tested and validated as Illumina ones.

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            • #7
              Originally posted by cheezemeister View Post
              I've been looking at doing some shotgun metagenomic sequencing on the HiSeq 2500 (or 3000/4000). It seems that with both Nextera DNA and Truseq Nano (or PCR-free) Illumina only has indexes that allow multiplexing up to 96 samples. I would, ideally, like to multiplex far beyond this, to 200 samples or more.
              Out of a matter on interest how many metagenomes do you intend putting on one lane? We are about to run some but I cannot seem to find a reliable answer as to how many reads are needed for good species level identification using programs like CONCONT and MetaPhlan. Obviously it will depend on the level of species present and if the samples have contaminating host DNA, as in from gut microbiomes, but any info appreciated.

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              • #8
                Bioo Scientific has launched 384 single-index barcoded adapters which are validated by sequencing. These barcodes contain a 12 nt index and are compatible with TruSeq style library prep, where the adapter is added during the ligation step of library prep.

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                • #9
                  Does anyone know of a commercial dual-10bp or longer index kit with at least 48 unique indexes for each end? Even 24 for each end would be better than what we currently use. For some ultra-low cross-contamination experiments we are running, every pair must be unique, no single barcode may be re-used, and single-end is insufficient; but we want to multiplex up to ~48 ways. Currently, we can only go up to 8 ways.

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                  • #10
                    Originally posted by Brian Bushnell View Post
                    Does anyone know of a commercial dual-10bp or longer index kit with at least 48 unique indexes for each end? Even 24 for each end would be better than what we currently use. For some ultra-low cross-contamination experiments we are running, every pair must be unique, no single barcode may be re-used, and single-end is insufficient; but we want to multiplex up to ~48 ways. Currently, we can only go up to 8 ways.
                    I don't know of one, but if you are using these in a rapid run, then you will want to use the cBot Duo kit (if you have a cBot...) Otherwise you will get bleed over from previous Rapid runs. It shouldn't be much, but by "ultra-low cross-contamination" I presume this might be an issue...

                    --
                    Phillip

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                    • #11
                      Originally posted by pmiguel View Post
                      I don't know of one, but if you are using these in a rapid run, then you will want to use the cBot Duo kit (if you have a cBot...) Otherwise you will get bleed over from previous Rapid runs. It shouldn't be much, but by "ultra-low cross-contamination" I presume this might be an issue...

                      --
                      Phillip
                      Thanks; I'll forward that to our libraries group. We do have a cBot but I don't know how it's operated.

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