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16S rRNA amplicon multiplexing on MiSeq

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  • 16S rRNA amplicon multiplexing on MiSeq

    How many samples can I run in a multiplex to get 'enough' reads per sample with an amplicon size of about 300 bp? Is there a formula to calculate this based on sequences generated per run, amplicon size and coverage?

  • #2
    Here is a coverage calculator from Illumina: http://support.illumina.com/download...alculator.ilmn There is a tech note included for the coverage calculation.

    An application note for 16s sequencing: http://res.illumina.com/documents/pr..._miseq_16s.pdf

    I will let someone else comment on what qualifies as "enough" reads.

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    • #3
      Thanks. The 'enough' questions is maybe more a good discussion point.

      Comment


      • #4
        Originally posted by wafi View Post
        How many samples can I run in a multiplex to get 'enough' reads per sample with an amplicon size of about 300 bp? Is there a formula to calculate this based on sequences generated per run, amplicon size and coverage?
        960 (10 plates of barcoded primers) using v2 2x150bp

        here's the protocol: http://www.earthmicrobiome.org/emp-s...protocols/16s/

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        • #5
          You can use the Coverage/Read calculator here: https://genohub.com/shop-by-next-gen...e7a8918f19251a I entered 960 samples at 30,000 reads/sample. Feel free to change those to fit your experiment.

          - Genohub
          Last edited by Genohub; 04-22-2014, 02:28 PM.

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          • #6
            What is the typical library concentration that people are using on the MiSeq? We are currently using 7 pM and getting ~800 k/mm2. Can we push this higher?

            Thanks

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