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Old 04-10-2014, 02:41 AM   #1
wafi
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Default 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?
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Old 04-10-2014, 03:01 AM   #2
GenoMax
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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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Old 04-10-2014, 03:20 AM   #3
wafi
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Thanks. The 'enough' questions is maybe more a good discussion point.
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Old 04-16-2014, 11:11 AM   #4
cement_head
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Quote:
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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Old 04-22-2014, 02:06 PM   #5
Genohub
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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 at 02:28 PM.
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Old 04-23-2014, 08:32 AM   #6
cement_head
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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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