An analysis aimed at addressing some questions generated following discussion of a previous post on GBS…

Number of fragments produced from a ‘digital digestion’ of the Nov22k22 Sunflower assembly:

Clai: 337,793
EcoRI: 449,770
SacI: 242,163
EcoT22I: 528,709
PstI: 129,993
SalI: 1,210,000
HpaII/MspI: 2,755,916

Here is the size distribution of those fragments (omitting fragments of >5000bp):
All the enzymes
With Msp removed for clarity

Take home message: PstI produces fewer fragments of an appropriate size than other enzymes. It looks like the correlation between total number of fragments and number that are the correct size is probably pretty high.

Now for a double digestion. Pst and Msp fragment sizes and again omitting fragments >5000bp. This looks good. In total fragment numbers (also omitting >5000bp fragments):

Pst+Msp total fragments: 187271
Pst+Msp 500<>300bp: 28930
Pst alone total: 79049
Pst alone 500<>300bp:6815

Take home: Two enzyme digestion could work really well. It may yield more than 4 times more usable fragments. I do think we could aim to get even more sites. Maybe some other RE combination could get us to the 100,000 range. With a couple of million reads per sample this could still yield (in an ideal world) 10x at each site. Send me more enzymes sequences and I can do more of the same.

Edited for clarity and changed the post name