Differences in read count from barcoded RADseq?

[susjoh]

Hello,

I have recently inherited some RAD data sequenced on an Illumina GAII platform. The protocol used was similar to Baird et al. (2008; see end of post for link). The samples were barcoded and sequenced in lanes containing libraries from two samples (each prepared sample should have had the same DNA concentration).

My question is related to the sequencing output. I have found a lot of variation between the read number for each individual sample; there can be up to a threefold difference in the number of reads between individuals.

Could anyone on this forum explain to me the reasons that there would be such large differences in read count from each sample?

Thank you!


Baird et al: http://www.plosone.org/article/info:...l.pone.0003376

[jboone123]

Do you know exactly how much variation you are seeing? Often times with next gen seq'ing projects variation between samples is quite common. If it is a tremendous difference I might be a little concerned but if it's not too much it shouldn't effect things too much.

If you are just curious about the reasons, here are a few:
1) different starting gDNA concentrations
2) different starting gDNA qualities
3) different starting primer/barcode concentrations
4) different starting primer/barcode qualities
5) PCR bias
6) Enzymatic digestion variance
7) sequencing variability (even multiplexed nextera samples are variable)
8) variable base calling (not likely)

Hope that helps!

Jason