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Old 10-19-2017, 08:02 AM   #1
Location: Scotland

Join Date: Apr 2015
Posts: 17
Default non-equimolar pooling to normalise read depth in libraries with different genome size

I'd like to sequence libraries of very different genome size on a single MiSeq run.

Eight bacterial samples with 4.5Mb genome size
One eukaryote sample with 23Mb genome size

Could I pool the libraries so that the larger eukaryotic sample represents 38% of the pool, rather than 11%? In theory, this would allow read depth to be the same across all nine libraries.
But what effect would it have on the MiSeq sequencing process if one barcode combination is present at x5 greater levels than the other eight?

Has anyone tried to do this, and did it work?

Thanks in advance
kerryp is offline   Reply With Quote
Old 10-19-2017, 09:49 AM   #2
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Location: CT

Join Date: Apr 2015
Posts: 243

The only concern is index diversity, Check your combinations to make sure that all channels are roughly equally represented across the indexes given that one index will be ~40% of the reads.
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 10-19-2017, 11:34 PM   #3
Location: Aarhus, Denmark

Join Date: Mar 2008
Posts: 57

Hi kerryp,
No problem. Have done it successfully for several years doing Nextera XT libraries of bacteria and sequencing on NextSeq medium in batches of 50-70 bacteria.
I havent had concerns about the indexes but as pointed out by thermophile you must give it some thought running only 9 samples.
Good luck,
JakobHedegaard is offline   Reply With Quote

genome size differences, indexing, miseq v2, nextera xt, pooling

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