Author Archives: Celine Caseys

How to do effective SPRI beads cleaning

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SPRI beads cleaning is one of the most repetitively used step during library preparations and probably the step where most of us lose a lot of precious DNAs.

Losing DNA scared me so much (because it can be observable) that I hesitated a lot before trying to use beads to concentrate genomic DNA, because usual rate of recovery are ~50%.

giphy

Hopefully with some practice and a lot of patience, it is possible to reach 90% recovery. How to lose as little DNA as possible? Here are some guidelines: Continue reading →

Phytochemical databases

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If you are interested by generating quickly a list of compounds that were previously identified in a plant, the online dictionary of natural product is a good place where to start your research. It regroups entries for >270k natural chemical compounds.

In the current version of the website (March 2016) you can search for “Biological source” and in there “the latin name of your favourite species”. The result of such a query for “Helianthus” is a list of 381 chemical compounds that were tagged as identified in Sunflower species (and a minority of other species with helianthus in the name). Of course this list is quite restricted compared to the number of metabolites produced by sunflower plants. It is however a good starting point to know what kind of compounds (here a lot of terpenes, few flavonoids) were previously extracted from your favourite plant species. Some entries are tissue specific, such as large terpenes from pollen. Also interesting, the “biological use” column gives sometimes information on the role of the compound (e.g. antifungal, allelochemical, growth inhibition).

The CAS registry number is probably the most powerful piece of information you can get out of such a search. It’s the unique ID for the compound and can be use to search the compound in multiple databases. My favourite are:

  • Chemspider: This is probably the more powerful chemical database that currently exists. It’s huge: it regroups information for more than 44 million chemical structure and cross-reference with many other databases.
  • Pubchem: It will look really familiar to NCBI/pubmed users. With pubchem you can for exemple submit the CAS number and get a list of publications linked to the compound.

 

 

 

 

 

How to do GBS libraries with “difficult” DNA samples

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First of all, let’s be clear about it: Having good amount of high quality DNA should be a starting point for all projects. Recently, we had this conversation at lab meeting about the “one rule” to succeed in establishing a lab (quoting Loren): “Don’t try to save money on DNA extraction. Working with high quality DNA reduces cost at all downstream steps, even on bioinformatics”.

However, if you need to work with “historical” DNA samples from the lab (I genotyped old DNA plates at least 8 years old) or DNA from collaborator for which you have no control over the DNA quality and/or no more plant tissue to redo DNA extraction, here are some tips on how to get a maximum out of (almost) nothing.

I started the GBS protocol with 100ng of DNA, it works. However, if you want to save yourself a lot of time and the lab some money on repeating PCR, repeating samples, repeating a lot of qubit measurement, start with 200ng.

A) If some of your DNA samples are <8.5 ng/ul (100ng protocol):

Among the 1500 DNA samples I received from a collaborator, 134 did not meet the requirement (>8.5 ng/ul) to start the GBS. I thought about concentrating these DNA with different methods: 1) using beads: you need to be ready to loose 50% of the DNA; 2) speedvac: I did not find one (supposedly there is one in the Adams lab?) and I was concerned about over-concentrating TE in the same time as DNA.

Hopefully, if you look at the digestion step, a large volume of the digestion mix is water/tris. By removing this water, I was able to include in the protocol DNA with concentration >5.8ng/ul, recovering half of my problematic samples. Just be extra-careful when pipetting the 2.8 ul of “water-free” digestion master mix. I had good PCR amplification for these samples.

B) If you are desperate:

I used whole genome amplification (WGA) prior to starting the GBS protocol to increase the DNA concentration of “historical” DNA samples. You will probably recover most of your DNA samples if they are more than 1ng/ul.

However, DO NOT MIX genome amplified and plain genomic DNA on the same plate for sequencing, especially if you pool your library before doing PCR and qubiting. The WGA samples amplify much better and Sariel showed me libraries in which few WGA samples took a large part of the sequencing reads. It’s a recipe for disaster and high missing data.

My strategy was to qubit all the DNA plates and estimate the remaining volume. If the remaining DNA sample was less than 100ng, I did WGA but I moved these samples to specific WGA plates. It’s a bit more work because if your samples are already in plates, you will need to relocate all your samples. From my experience, it’s worth it.

Whole Genome Amplification

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I used Whole Genome Amplification (WGA) to recover some very old DNA and include a maximum of samples into GBS plates for a mapping project. I will do another post about that, but here I would like to resume how I did the whole genome amplification.

For information on how the whole genome amplification method works, check the previous post by Moira.

I used the Qiagen Repli-g Mini Kits with the amounts divided by half.

The protocol is the following:

2.5 ul of DNA

2.5ul of buffer D1 (DLB + nuclease-free H2O)

Incubate for 3min at room temperature

Add 5ul of buffer N1 (stop solution + nuclease-free H2O)

Vortex and centrifuge

Add 15ul of master mix (repli-f reaction buffer + DNA polymerase)

Incubate at 30oC for up to 16h and inactivate enzymes at 65oC

I found that 6h was good for the amount of DNA I was hoping to recover. I recovered at least 50x the amount I started with, typically starting with concentrations between 1-10ng/ul and getting on average 60ng/ul out of the WGA protocol.

Depending on the use of the DNA, you may want to play with the incubation time (e.g. keep it short), especially if you plan to use PCR based approach later on (almost everything is PCR-based..).