How to collect for "Sample collection for metabarcoding"

jeudi 4 juin 2015
par  Aurélien DE JODE, Dorian GUILLEMAIN, Romain DAVID
popularité : 3%

Index of "Sample collection for metabarcoding"
1. Principle
2. Method
3. Sampling
4. Deliverables
5. To summarize
Back to protocols Index
How to cite this content :

David R., Arvanitidis C., Çinar M.E., Dubar J., Dubois S., Erga Z., Guillemain D., Sartoretto S., Thierry de Ville d’Avray L., Zuberer F., Chenuil A., Féral J.-P., (2014), with contributors : Açik Çinar S., Andral B., Aurelle D., Aysel V., Bakir K., Bellan G., Bellan-Santini D., Bouchoucha M., Celik C., Chatzigeorgiou G., Chatzinikolaou E., Chenesseau S., Dağli E., Dailianis T., Dimitriadis C., D’Iribarne C., Doğan A., Dounas C., Egea E., Elguerrabi W., Emery E., Evcen A., Faulwetter S., Gatti G., Gerovasileiou V., Güçver S.M., Issaris Y., Katağan T., Keklikoglou K., Kirkim F., Koçak F., Koutsoubas D., Marschal C., Önen M., Önen S., Öztürk B., Panayiotidis P., Pavloudi C., Pergent G., Pergent-Martini C., Poursanidis D., Ravel C., Reizopoulou S., Rocher C., Ruiton S., Sakher S., Salomidi M., Sarropoulou E., Selva M., Sini M., Sourbes L., Simboura N., Taşkin E., Vacelet J., Valavanis V., Vasileiadou A., Verlaque M. Protocols for monitoring of coralligenous habitats of mediterannean (Coralligenous based Indicators to Evaluate and Monitor the "good ecological status" of the MEDiterranean coastal waters) Protocoles de suivi du coralligène en méditerranée (Coralligenous
based Indicators to Evaluate and Monitor the "good ecological status" of the MEDiterranean coastal waters).

a. Operations

[§ 1] For sampling, it is necessary to use a succion sampler (Figure 1) connected to a scuba tank.(Figure 2).

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Figure 1 : succion sampler

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Figure 2 : scuba tank for suction sampler

[§ 2] Undewater, the succion sampler should be placed above the scraping area (Figure 3).

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Figure 3 : scraping and aspiration by succion sampler

[§ 3] The sample rises in the PVC tube and is retained by the net on the top of succion sampler (Figure 4).

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Figure 4 : the net on the top of succion sampler

[§ 4] At the end of scraping, the net is untied fastened (tiing a knot at the open extremity of it) and placed in a zip bag (Figure 5).

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Figure 5 : the net is put in zip bag

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Figure 6 : the four scrappings are completed

[§ 5] On the boat, Check that the water quantity allowed in the bags is sufficient (complete if necessary) and put samples in the cooler (Figure 7).

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Figure 7 : sample in the cooler

Be careful :

If the scuba tank of the succion sampler empties completely during sampling, sea water can enter inside because of the pressure difference. If this happens, the tank deteriorates rapidly and needs to be inspected by a certified company.

In order to prevent sea water intrusion into the tank, is highly recommended to equip it with a manometer and to check remaining air after each sample.

b. Back to the lab
[§ 6]

There are two ways to proceed depending the time and the human ressources you have at the lab at the arrival of the samples. What is important to keep in mind is that all the samples that are relevant to compare must be processed the same way.
A) If you have sufficient time at the arrival of the samples :

1) The content of each scraping is spread in a white tray with sea water, in a fresh and well lightened place, to be rapidly sorted until conditioning in ethanol 95°. (for label coding see below).

2) Take a picture of the sample, zooming when necessary, allowing (possible) a posteriori identification. Samples and photographs must be labelled as follows :
[program]_[site? ?]_[date] _D[depth]_[profil? ?]_[position] _[doer] Exemple : CIGESMED_CAS_20140121 _D1_V_TOP_AA01

3) Try to determine the number of visible species per phylum (though rapidly, since each dive provides usually 16-32 units to process).

4) Separate as much as possible the bioconstructed coralligenous, brush with a hard tooth brush in the tray seawater.

5) Proceed to fractioning using the sieves (3 fractions as in the DEVOTES WP5 protocol). Wear gloves :
a. Fraction > 2mm
b. Fraction between 2 mm and 0,5 mm
c. Inferior fraction (inferior to 0,5 mm) : collect with an ethanol dropping bottle what remained on the sieve

6) Each fraction is preserved in ethanol 95° at 4°C waiting for molecular analysis.

B) If you have few time :
1) Keep the samples in the stocking and dry it the best you can by pressing it on your hand (gloves).
2) Place the stocking in a small bottle full of 95° ethanol and place the bottle (Figure 8) at 4°C.
3) The day after, collect the ethanol contained in the bottle and in the sctocking by pressing it in your hand. Put this « old » ethanol in another bottle and be sure that it is labelled properly (may be used for micro-organism diversity investigation).
4) Put the stocking back in its small bottle and refill it with 95° ethanol.
5) Conserve all the bottles at 4°C .
6) When you have sufficient time to proceed to the fractioning start the protocol at step 2 of the part « A) if you have time at the arrival of the samples ».

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Figure 8 : Bottle used to store the samples

[§ 7]
Field material : 
- Dive weight with 1 m rope (or thread) 
- 10cm x 10 cm quadrat frames 
-  Scraping tool 
- Succion sampler 
-  Nets 
-  Pre-labelled plastic zip bags
- Conditioning/packaging material : 
-  Ethanol 95% (around 4-5 liters for a set of 4 scrapings of 10cm2)
-  White trays (size > A4) 
-  2 Sieves (2 mm and 0,5 mm) 
-  Dropping bottles 
- Bottles 500 ml (one for each replicate)

d. Work time quantification
[§ 8] Positioning takes about 3-5 minutes, then each scraping takes about 1-3 minutes. Finding a new location for the central point, corresponding to the requested ecological profile?, about 1-2 minutes (it is important to take the time to find an ecologically homogeneous profile). Each diver -team (2 persons)- can thus obtain about 4 scrapings (1 quadriplicate) in one dive.

[§ 9] Amount of diving-work per site : 2 profiles X 2 orientations X 2 depths x 4 replicates = 32 scrapings on 3 sites minimum, up to 8 ideally).

[§ 10] The time requested for molecular analyses depends on the number of phyla or classes (i.e. the number of primer pairs) to be amplified by PCR. Those analyses are performed for large sets of samples (it is worth starting them when 32 scrapings are ready, so it makes 96 DNA extractions, one for each fraction, 1 day work-time). Once all DNA are extracted, PCR amplification is automatized by series of 96 samples and 2 x 96 PCR and the corresponding agarose control gels are easily performed in a day. Then samples must be sent for sequencing to a NGS platform.


- Deadline : 14th December 2015
- Please add comments for each paragraph (§) by means of a corrected new version (one form per §)
- It is recommended to add scientific references if necessary

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