Experimental protocol 2: Assessment of structure of prokaryotic communities by
454-sequencing
Chemicals and Reagents
NucleoSpin Soil DNA kit (Macherey-Nagel), NucleoSpin Plant II kit (MachereyNagel), isopropanol, ethanol, sterile deionized water, eub530F primer (5´GTGCCAGCMGCNGCGG - 3´, 10 µM), eub1100aR primer (5´GGGTTNCGNTCGTTGCG - 3´, 10 µM), Pfu DNA polymerase (Fermentas,
Hannover, USA), DyNAZyme II DNA polymerase (Finnzymes, Espoo, Finland), 10x
buffer supplied with DyNAZyme II DNA polymerase, 1% solution of bovine serum
albumin, 10 mM PCR nucleotide mix, tagged eub530F primers with Titanium A
adaptor, eub1100aR primer with Titanium B adaptor (as described in Baldrian et al.,
2011), SYBRgreen, agarose, Wizard SV Gel and PCR Clean-up System (Promega),
MinElute PCR Purification Kit (Qiagen), Quant-iT™ PicoGreen ® dsDNA Assay
(Invitrogen), emulsion PCR GS Junior Titanium emPCR Kit Lib-L (Roche), GS Junior
Titanium PicoTiterPlate Kit (Roche) and GS Junior Titanium Sequencing Kit (Roche).
Material
Autoclave, thermocycler, Nanodrop spectrophotometer, centrifuges, gel
electrophoresis, microplate reader (UV-VIS range), pipettors, pipette tips, microtubes,
centrifugation vials, GS Junior System (454-sequencing, Roche)
Method protocol
1. Extract DNA from soil or ectomycorrhizae samples (250 or 100 mg fresh
weight, respectively) with the respective DNA extraction kits following
manufacturer´s recommendations. Assess concentration and quality of DNA
with Nanodrop.
2. Amplify the V4-V6 region of 16S rDNA with non-tagged primers
eub530F/eub1100aR. Each sample shall be processed in three analytical
replicates. Each reaction (50 µl) shall contain:
a. 0.12 U Pfu DNA polymerase
b. 3 U DyNAZyme II DNA polymerase
c. 5 µl of 10x buffer supplied with DyNAZyme II DNA polymerase
d. 3 µl of 1% bovine serum albumin
e. 1 µl of 10 mM PCR nucleotide mix
f. 2 µl primer eub530F (10 µM)
g. 2 µl primer eub1100aR (10 µM)
h. 12 ng template DNA
i.
water up to 50 µl
Final concentration of Mg2+ shall be 1.5 mM.
Cycling conditions shall be:
Initial denaturation at 94 ºC for 5 min
35 cycles of 94 ºC for 1 min, 62 ºC for 50 s, and 72 ºC for 30 s
Final elongation at 72 ºC for 10 min
3. Pool the three analytical replicates of the previous PCR step
4. Purify the DNA with isopropanol precipitation and redissolve in water
5. Carry out a second amplification step (two analytical replicates per sample)
under the same conditions as above. Use tagged eub530F primer with
Titanium A adaptor and eub1100aR primer with Titanium B adaptor (Roche,
Basel, Switzerland) as described in Baldrian et al. (2011). Use only 10 cycles
and 200 ng DNA template per reaction.
6. Pool the product of the replicate PCR reaction from the previous step, stain
the samples with SYBRgreen and separate on 2% agarose gel.
7. Cut off the DNA fragments within the range of 400-1000 bp, extract DNA with
Wizard SV Gel and PCR Clean-up System (Promega) and purify with MinElute
PCR Purification Kit (Qiagen).
8. Measure concentration of purified DNA with PicoGreen
9. Produce equimolar mixture of all purified DNA samples for the 454-sequencing
10. Subject the sample to the emulsion PCR using GS Junior Titanium emPCR Kit
Lib-L and sequencing on the GS Junior platform.
Data analysis
1. Sort the sequences obtained by 454-sequencing according to their tags
2. Check for the presence of chimaeras using the tool Uchime (a component of
the software package Usearch, http://www.drive5.com/usearch). Remove
chimaeras from the dataset
3. Remove sequences shorter than 500 bp
4. Cluster the remaining sequences using the tool CD-Hit (http://weizhonglab.ucsd.edu/cdhit_suite) at 95% level of similarity.
5. Find the closest identified counterpart for each sequence cluster (operational
taxonomical unit, OTU) in the Ribosomal Database Project
(http://rdp.cme.msu.edu).
6. Discard chloroplast sequences
7. Merge OTUs with the same identification
8. Discard OTUs identified in 5 or less samples (out of the 30 samples in total)
Reference
Baldrian, P., Kolařík, M., Štursová, M., Kopecký, J., Valášková, V., Větrovský, T., et
al. (2011) Active and total microbial communities in forest soil are largely different
and highly stratified during decomposition. ISME J., doi: 10.1038/ismej.2011.95