Tag Archives: restriction enzyme

Where does all the GBS data go? Pt. 2

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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

PstI digest tests

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Edit: I forgot to thank DanB and Kate for the generous donation of DNA.  Sorry guys 🙁

Edit 2: After the lab meeting some questions have been asked.

To summarize :

1.  All the enzymes work equally well.  There is a slight performance decrease if one uses the PstI-HF.  

2.  I’d recommend using the PstI from Invitrogen – Invitrogen works extremely well and is the cheapest among the Psts tested ( $22 versus $75  ).  EDIT: Brook and Kate let me know that I failed to factor in the units provided for a given dollar value.  Invitrogen provides the cheapest enzyme.

For NEB – With our volume purchases we get 10000U for $71.40.

For Invitrogen – With our volume purchases we get 10000U for $63.90 {represents price negotiated with Helen, accounts manager at Invitrogen Stores}

Thanks guys for pointing that out!

This adds up considering how much we’re going through.

3. Sunflower gDNA does not digest completely in 3 hours.  It is recommended to go overnight or 18 hours with your digestion.

4. After 18 hours do not be alarmed by incomplete digestion.  This is OK according to RFLP work performed by Loren.  There should be a sufficient number of fragments for GBS libraries.

Hi all,

Here are the results from the PstI digest tests:

All enzymes tested fail to fully digest sunflower gDNA.

Enzymes from Invitrogen, Thermofisher/fermentas, NEB (non HF) and NEB HF were tested.  All performed equivalently.  I would say the NEB HF was slightly less processive after 70 min.  See attached PDF for gels and full documentation of reaction conditions.

**** 2013-Oct-08-debono-digesttest *****

Thanks

Allan