SEQanswers Why does number of reads per sample vary?
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 03-28-2018, 11:38 AM #1 MD19 Junior Member   Location: usa Join Date: Jan 2017 Posts: 2 Why does number of reads per sample vary? Hi all, I am just starting to learn NGS data analysis.So this may be a very basic question. So in the data for your samples which were normalized in quantity for sequencing run which are expected to have say 10 million reads each, what does it mean if you get a Gaussian distribution averaging at 10million but with +/- 3 million reads? how do you explain that? Thanks
 03-29-2018, 03:20 AM #2 Bukowski Senior Member   Location: Aberdeen, Scotland Join Date: Jan 2010 Posts: 347 Well - you try pipetting so accurately that you can manage better... Quantification isn't exact. Dilution isn't exact. Pooling isn't exact.
03-29-2018, 08:21 AM   #3
MD19
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Thanks, Bukowski! Could it also be 'quality of the sample' issue affecting the final output? Just trying to think of all possibilities in this scenario.

Quote:
 Originally Posted by Bukowski Well - you try pipetting so accurately that you can manage better... Quantification isn't exact. Dilution isn't exact. Pooling isn't exact.

 03-29-2018, 10:32 AM #4 SNPsaurus Registered Vendor   Location: Eugene, OR Join Date: May 2013 Posts: 435 Sure, fragmented DNA may make libraries that have a shorter fragment length. Unless you are precise in figuring out how many fragments are in your sample's library, the normalization will be off (and it is a given that you won't be precise at this since fragments can tangle and daisy chain which affects the apparent size distribution. If they are ever pooled and amplified, the short fragments will be enriched in the pool as well. __________________ Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

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