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Old 11-04-2013, 09:56 AM   #1
frossit
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Default Multiplexing human microdissected chromosomes with human whole genome

Hi !!!
I would like to know if i can multiplex a single human microdissected chromosome with whole human genome in a single lane of a Illumina 2500 flowcell ? Coverage of the single human chromosome should be arround 50 X. We are using the Truseq PCR free for library prepair.
Thanks
Fabio
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Old 11-04-2013, 10:43 AM   #2
dpryan
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Which chromosome (they kind of vary a lot in size...) and what read lengths are you going to use? Or do you need us to figure that out for you?
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Old 11-04-2013, 11:07 AM   #3
frossit
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Thanks for your answer !!! I´m trying to help a friend of mine. I will collect more informations and post them here....
Best wishes
Fábio
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Old 11-05-2013, 09:40 AM   #4
frossit
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The microdissected chromosome has 160 megabases. We are planning 2x75bp with 50X coverture sequencing protocol in a Illumina Hiseq 2500 . Can I multiplex this chr with a whole human genome in a single lane of the flowcell ? If is it possible, coverture of the whole human genome have to be exacty the same (50X) or i can sequence this genome in the same lane but with a differente coverture (for example..10X for the genome and 50x for the chr) ?
Thanks
Best Regards
Fabio
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Old 11-05-2013, 12:48 PM   #5
dpryan
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You'd need about 60 million reads (I'm assuming ~90% alignment and that the reads in a pair don't overlap) for 50x coverage of that chromosome. That should be doable in 1/4-1/3rd of a lane (check with whomever is running it, they'll have a better feel for how much useful data they can reliably get out of a lane). Assuming maybe 200 million total reads (likely more, but let's be conservative), then the remaining 140 million reads would maybe produce an approximate 9x or so coverage in an ideal world, though likely less in practice. Again, chat with whomever is running the machine that will handle these and they can give you a better idea of what you can expect to get.
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