LIFE08 ENV/EE/000358
Guidelines and user manuals for monitoring the
environment through the status of the soil microbiome
Table of contest
1. Introduction
1.1. Background
1.2. General methodical approach and the application workflow
1.3. Output
2. Methodical approaches
2.1. Sample collection and pre-analysis handling
2.2. Indicator species determination
2.3. Guidelines for primer design and selection for qPCR
2.4. qPCR guidelines
3. Determination of the score of anthropogenic pressure (Pscore)
4. Assessment of the environment according to the monitoring results
5. Appendices
Appendix I Manual 1. Collection of soil samples for metageneomic analysis of
microbiome diversity.
Appendix II Manual 2. Storage of soil samples over a longer period.
Appendix III Manual 3. Preparation of soil samples for DNA purification.
Appendix IV Manual 4. Purification of total DNA from soil samples
Appendix V Table 1. Primer sets for the BIOTAGENE method
1. Introduction
1.1. Background
Current guidelines and user manuals for BIOTAGENE method are a result of a LIFE+
Project LIFE08 ENV/EE/000258 “Elaboration of novel metageneomic method for
environmental monitoring” carried out at Tallinn University of Technology Centre for
Biology of Integrated Systems (CBIS) between January 2010 and July 2012. The
project has been a further development of the research activities carried out at CBIS
since 2007, dealing with the application of metageneomic approaches upon
environmental monitoring. The aim of the activities under the current project was to
elaborate a generally applicable cost-effective methodical approach usable in
environmental screening to evaluate the degree of anthropogenic pressure based on
metageneomic analysis of the corresponding changes in the community of soil
microorganisms. The starting point in the BIOTAGENE method development has
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been the general approach of identification and quantification of the members of the
soil microbiome by means of high through put highly parallel 454 pyrosequncing
allowing to collect corresponding information for thousands of the community
members at a time. It is quite clear that although this kind of an approach provides the
most detailed and informative data achievable, it cannot be feasible in everyday
environmental monitoring, due to high costs and labor intensity. Guide by the
mentioned concerns a practical methodical real-time PCR based approach for
identification and quantification of indicator/marker species in the soil microbiome
permitting assessment of the environment has been elaborated and verified on a set of
real environmental samples gathered from 409 locations over the period of the project
span. The method’s out put is a score of anthropogenic pressure (from 0 to 1 on
the scale) upon given environment derived from mathematical modeling of the
metagenomic data collected and the RT-PCR method development. In the current
version the scores are calculated based on the RT-PCR results provided by the defined
minimal set of 16 indicator species, each of which carries its share in environment
assessment.
1.2. General methodical approach and the application workflow
The current version of the BIOTAGENE method developed serves as a first proof of
principle for the applicability of the results of the soil microbiome metagenomic
research performed at the Centre for Biology of Integrated Systems (CBIS), Tallinn
University of Technology (TUT), over a period more than half a decade, for
environmental monitoring.
The BIOTAGENE method can be described as a package of diverse activities,
methods and approaches, originating from miscellaneous fields of biology-connected
disciplines allowing to describe the environmental status based upon the soil
microbiome. For the development of the BIOTAGENE methodology expertise and
academic knowledge from the fields of soil science, ecology, microbiology,
mycology, molecular biology, biostatistics, bioinformatics and biotechnology has
been utilized resulting in an interdisciplinary approach for the assessment of
surrounding environments. The assessment of environment according to the
BIOTAGENE method is carried out according to the presence, absence or relative
representation of the indicator species. The last have been identified as a result of
analyzing soil probes collected from 409 sampling sites in countries surrounding the
Baltic Sea (Estonia, Latvia, Lithuania, Poland Germany, Sweden and Finland)
characterized as varying in the level of anthropogenic pressure. For all the collected
soil samples metagenomic analysis by characterization of the soil microbiome
according to specific regions of the 16S ribosomal RNA (for Bacteria and Archaea)
was carried out by means of 454 high throughput pyrosequencing. The resulting
sequencing data was used in pattern analysis. The representation patterns of the
species in the samples served as bases for selecting the indicator species and the
corresponding DNA sequences were used for the specific primer design. The
designed primers were used as an input for developing the qPCR protocols serving as
a backbone for data generation in the developed prototype method.
The general workflow chart of the method (Fig.1) assists in understanding the
approach and helps planning the sequence of activities to be carried out. The
workflow can be divided into three major parts. The first of them deals with sample
collection, handling and archiving. Detailed descriptions of the assisting actions are
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given in more detail in the accompanying manuals. Manual 1 (Collection of soil
samples for metageneomic analysis of
microbiome diversity) gives guidelines for
fieldwork and its preparatory activities.
While Manual 2 (Storage of soil samples
over a longer period) deals with the issues
and requirements of sample archiving.
The second set of sub-activities involves
preparatory actions for and laboratory work
involving molecular biological methods to be
sequentially applied upon the collected
samples. In detail the guidelines and
protocols are given in Manual 3 (Preparation
of soil samples for DNA purification),
Manual 4 (Purification of total DNA from
soil samples) and in the current document
(qPCR guidelines).
The third set of activities named on the chart
as “Determination of the Pscore” and
“Monitoring results” involves analysis of the
data produced by the workflow and
formulation of the assessment of the
environment based upon it. The guidelines
for these activities will be given in the final
parts of the given user manual.
One should keep in mind, while using this
manual and the accompanying protocols, that
we are dealing with a prototype and that we
provide the guidelines, which have been
working for us. The manuals are meant for
any interested parties to be adapted to their
own conditions, technical capabilities and
aims. On the other hand while redesigning
the details of protocols one should keep in
mind that all the steps in the workflow are
interconnected and one should try to avoid
principal changes on any step of the
workflow as not to influence outcome.
1.3. Output
Based upon qPCR results the value of the
score of anthropogenic pressure (Pscore)
upon the given environment is calculated.
The scale of the score spans from 1 (the least
anthropogenic pressure) to 0 (the highest anthropogenic pressure). The calibration of
the scale is based on the 5 step ecosystem’s metadata connected gradation in use at
the University of Tartu Frontiers in Biodiversity Research (FIBIR) Centre of
Excellence. To give the understanding of the biological meaning of the Pscore we
hereby give a descriptive list (Table 2) of environments corresponding to the scores.
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Table 2.
FIBIR BIOTAGENE score of
grades anthropogenic pressure
(Pscore) intervals
5
0.8 – 1
4
0.66 – 0.8
3
0,41 - 0,65
2
1
0.16 – 0.4
0 – 0.15
Ecosystems characteristic to the intervals
Primeval forest, non managed growing forest,
coastal meadows with low mowing or grazing
pressure, etc.
Natural grasslands with intensive mowing or
grazing pressure, managed forests subjected to
thinning, suburban forest parks, other recreation
areas with natural vegetation, etc.
Cultivated pastures, fallow land, intensively
used recreational areas, city parks etc.
Agricultural fields, etc.
Mining spoil tips, areas submitted to intense
landscaping, city lawns in high air pollution and
intensive treading areas, etc
The scores are a measure of anthropogenic pressure compared to untouched nature.
The scores in themselves describe the status of the ecosystem only at the moment of
sample collection. For monitoring purposes the scores have to be compared between
samples taken from the same collection area over intervals of time, as to evaluate the
deterioration of the status or the remediation of the environment, for example after
recultivation of mining areas, etc. Based on the Pscore calculated the environmental
assessment can be carried out.
2. Methodical approaches
2.1. Sample collection and pre-analysis handling
The fieldwork for sample collection starts with the planning phase and preparatory
activities. The first step is to collect information about the sample collection area and
get hold of as much preliminary metadata as possible. In that sense different Internet
services, best known of which is Google Earth, are of great help. Also one has to go
through the supplies needed for field work and see that all the needed appliances are
in good order and in sufficient amounts. It is suggested to think the sample colletion
procedure through as thoroughly as possible and accordingly prepare sufficient
amounts of sample collection containers, labeled accordingly. Also sufficient
documentation should be prepared and all the possible changes in the plans during the
fieldwork due to possible insufficient preliminary information should be thought
through. The details of the sample collection and general guidelines for post
collection sample handling are given in Manual 1 and Manual 2, which are suggested
to be worked through properly before taking any action, including the planning phase.
The work in the lab starts the very moment the sample the samples are passed through
the doors of the premises. One has to take care that the soil in the samples will dry in
a shortest time possible and will stay the way during the whole storage period. That is
of paramount importance as the lack of water in the samples is the factor inhibiting
the bacterial reproduction and biological activities, which otherwise might cause
significant changes in samples’ DNA profile compared to the conditions in the field
the moment the samples were collected. The lack of water traces in the sample could
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be seen as a factor fixing the status of the microbiome to the moment of collection. It
is expected to be taken care of by the dry silica gel already present in the sample
collection containers. But it should be kept in mind that weather conditions are never
perfect and the same and attention should be paid to the humidity conditions in the
containers. One should follow up the status of the samples during the subsequent to
fieldwork days and add extra silica gel to the sample collection containers until one
can be sure enough the humidity challenge in the samples has been taken care of.
We do not give detailed guidelines concerning the sample archive documentation
setup as it should conform to the general routines of every specific working
environment, but would like to emphasize here that it is of utter importance to take
time and think it through even before starting the field work, as to avoid mix-up of the
samples when they start accumulating to avoid piling up during the later stages of
research activities. One should take special care to keep the samples linked with the
collected before, during and after the fieldwork metadata and other documentation,
which usually is of vital importance when interpreting the results during the
environment assessment.
The sample preparation for DNA purification step can be observed as an intermediate
step between the pre-analysis and analysis steps in the workflow as the samples can
be kept archived for extended periods even after being packed with the
homogenization beads into homogenization tubes. A detailed protocol of hands on
activities of preparation of samples for homogenization is given in Manual 3
including the guidelines of precautions to be taken during the procedure as to
minimize the threats of introducing artifacts into the samples during the handling
steps.
The last step of preparatory activities before the actual analysis is the purification of
DNA from soil. A sufficiently detailed protocol for the procedure is provided in
Manual 4.
2.2. Indicator species determination
For indicator species determination several multiparametric statistical methods were
implemented upon the metagenomic data generated by 454 sequencing. The principal
component analysis (PCA) was the best to identified the differences between the
samples collected from areas with varying levels of anthropogenic pressure applied.
The method together with the principle coordinates analysis (PCoA) enabled
demonstration of well defined microorganism communities in different biotopes. The
PCA performed took into account about 60 000 dimensions provided by the dataset,
the number corresponding to the amount of OTUs identified throughout all the
samples studied. The results of PCA formed the basis for selection and modeling of
site-specific indicator taxons. For these purposes modeling by means of glmnet was
performed. For glmnet two pools of samples were selected. The ones with the 2nd PC
(principle component) value between -0,02 and -0,04 (corresponding to the pristine
environments) and affected environmental samples with PC value >0,02. The model
resulted with the selection of 134 OTUs with presumed diagnostic value for the
determination of environmental disturbance. Of these 134 a minimal set of 16 OTUs
was defined carrying the biggest share of specificity information in defining the level
of anthropogenic pressure upon the environments.
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2.3. Guidelines for primer design and selection for qPCR
Primer design is a central objective for making the BIOTAGENE methodology
operational for assessment of environment. All the method is built around the
presumption that it is possible to identify indicator species of microorganisms, which
has proven to be correct. The indicator species or operational taxonomic units (OTUs)
are identifiable through the differences in their ribosomal DNA’s variable regions.
The exploitation of the high throughput 454 sequencing approach generated enough
DNA data (up to 5000 sequences per sample) to pinpoint, through the multiple
component and pattern analyses the species allowing to differentiate between
environments defined by the level of anthropogenic impact. Based on the list of
indicator species their available in the Gene Bank ribosomal genomic sequences
served as an input for the design of primer sets. The primers were designed using the
developed in CBIS proprietary primer design algorithm and verified by means of the
Primer3 and Primer Express software packages. While designing a set of multiple
primer pairs to be used in parallel in one qPCR run on one PCR plate the general
precautions for such procedures are taken. The primer pairs admitted for the final set
and usage in practice must meet the basic requirements like: the primers’ specificity
must be high, the melting temperatures of all primers must be as close as possible, the
amplicon lengths of the PCR products must be of a comparable size, primer dimere
formationshould be avoided. According to these basic demands while choosing the
primer set sequences the entire available ribosomal genomic regions were used as a
target. In the current BIOTAGENE version the aim was to design primer sets where
the forward and the reverse primers as well as TaqMan probes all add to the
specificity of the amplification reaction. Such an approach allows implementing the
tests on different methodical platforms. Using all the three primers in one reaction is
resulting in the highest specificity using the TaqMan approach. At the same time
using the forward and reverse primers alone gives adds an option to use the SYBR
Green I method, significantly decreasing the overall costs, but the same time prone to
not separate between the specific and unspecific amplicons. On the other hand the
primer pairs with high specificity allow an option also to use the method when one is
lacking high price equipment like RT-PCR machines and instead visualize the
amplication products by regular gel electrophoresis. It should be stated here that yet
the method of choice is the exploitation of TaqMan probes producing the results most
straight forward for interpretation while the SYBR Green I method and visualization
by electrophoresis add subjectivity and thus ambiguities to the assessment of the
environment although lowering significantly the costs of the undertaking.
The list of primer sets evaluated for the current prototype version of the Biotagene
method are given in Table 1.
2.4. qPCR guidelines
The details of the methodical setup of the qPCR step of the workflow depend from the
equipment available and the laboratory routines in use. Here we give a description of
the mentioned part of the workflow in use at the CBIS and some practical hints
helping to streamline lab-work and lower the running costs.
The current version of BIOTAGENE method has been developed using
StepOnePlus™ Real-Time PCR System. For TaqMan probe reaction we used the
TaqMan® Universal PCR Master Mix while adjusting the reaction volumes to 20 l
and following the guidelines given in the manual. The melting temperatures were
adjusted by using the temperature gradient function available on the StepOnePlus
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system.
The primer sets were also evaluated for the usability while exploiting the SYBR
Green I technology. In our experience a significant cost decrease in using the SYBR
Green I method could be achieved while instead of the commercially available
corresponding master-mix one uses an in-house compiled master-mix. Beside cost
effectiveness this kind of approach adds flexibility in modifying conditions to
improve reaction efficiency and specificity. Also the option of using the Taq
polymerase of one’s choice is introduced.
At CBIS we have taken advantage of using a high concentration (25 U/l) Taq
polymerase and the example of setting up SYBR Green one reaction based on in-hous
made master-mix is compiled accordingly.
The list of chemicals needed is as follows:
MgCl2
100 mM stock solution
dNTP
10 mM stock solution
200 M final concentration
Taq polymerase
25 U/l
0.1125 U/l final concentration
10x standard reaction buffer with MgCl2 (final conc. 3,5 mM)
10X reaction buffer without MgCl2
SYBR Green I gel stain (http://products.invitrogen.com/ivgn/product/S7563)
provided in 10,000x concentrate. Dilute this stock first to 100x in 10 mM Tris, pH 7.6
(i.e. 200 times). Use at 0.5 x
6-Carboxy-X-rhodamine is supplied as powder. Dissolve in dimethylformamide to 6
mM (dissolve 10 mg in original vial with 3.1 ml DMF).
Dilute this stock to a working solution of 60 µM in DMF. Use at a final conc. of 1.87
µM (1 ng/µl).
As to assist and simplify mixing together a sufficient amount of master-mix for the
desired amount of reactions we have constructed an Excel based support tool as
demonstrated on Fig.2. On the given snapshot guidelines of making enough mastermix for 96 reactions at different MgCl2 concentrations is displayed. In the orange cells
up left one can change the reaction number and the volume. The number for 100
reactions warranties that due to possible pipeting discrepancies enough master-mix is
made for 96 reactions. The lowest line of cells display the volume of master-mix to be
introduced to a feeder plate well as to use an eight channel pipet when transferring the
mix to the reaction plate. The PCRs are set up the way that 10 l of master-mix is
mixed in the reaction with 2 l of primers and 8 l of template resulting in 20 l
reactions. CBIS is ready to share the master-mix calculator Excel file upon request.
It has to be pointed out here that while setting up the in-house master-mix
approach one has to optimize and verify its compatibility with the equipment
available and the Taq polymerase and reaction buffer used.
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3. Determination of the score of anthropogenic pressure (Pscore)
Resulting the qPCR procedures one gets 16 Ct values (y1,…y16) for every sample
measured. From these values the sample’s score of anthropogenic pressure (Pscore) is
calculated.
As a first step in the calculations one has to estimate xi (the proportion of the given
indicator species i in the given sample) in correspondence with the following formula,
xi  c
1
n
1.6
1.6yi
y j
j 1
where coefficient c is the sum of relative abundances of all the 16 selected indicator
 by metageneomic studies dataset. In the given version* of the
species in the perused
BIOTAGENE method the value of c=0.02 in all the cases; yi is the Ct value of the
given indicator species in the given sample where i=1,...,16; j=1,…, 16. The constant
1.6 is the average amplification efficiency value characteristic for the 16 primer sets
used at the CBIS facilities at the current reaction conditions. Every user should
estimate that value for his own conditions.
*in the next version of the method it will be substituted with the sum of relative abundances of all the indicatorspecies in the
given sample by intrducing a primer set corresponding to the amplification of all the bacterial 16S genomic DNA independent of
the species specificity allowing to calculate a specific c value for every sample based on qPCR procedures.
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Based on the xi values calculated, the next step is to determine the g value (the
unscaled value of the score of anthropogenic pressure (Pscore)) according to the
formula
where i is the “purity” coefficient for the given (i=1….16) indicator species allocate
by the glmnet model. The corresponding values for every indicator are given in Table
3. The final scaling of the score of anthropogenic pressure (Pscore) is carried out
according to the formula
or alternatively expressed as follows
Pscore=1/(1+e-g)
The resulting scaled values of Pscore for the samples measured should fall into the
interval between 0 and 1.
As to get the touch of the meaning of the Pscore assigned to a specific sample please
refer to Table 2 and section 1.3 of the current document.
Table 3.
Indicator #
INDICATOR 1
INDICATOR 2
INDICATOR 3
INDICATOR 4
INDICATOR 5
INDICATOR 6
INDICATOR 7
INDICATOR 8
INDICATOR 9
INDICATOR 10
INDICATOR 11
INDICATOR 12
INDICATOR 13
INDICATOR 14
INDICATOR 15
INDICATOR 16
GLMnet
“purity”
coefficient i
-213,41
-148,33
-143,41
-135.89
-125,31
-117.32
-115,89
-107,77
39,81
42,98
45,8
46,52
74,54
98,78
106,17
390,05
Characteristic of
environment
disturbed/undisturbed
disturbed
disturbed
disturbed
disturbed
disturbed
disturbed
disturbed
disturbed
undisturbed
undisturbed
undisturbed
undisturbed
undisturbed
undisturbed
undisturbed
undisturbed
Interested parties are welcome to download an Excel based Pscore calculator from the
project’s website http://moonfish.ttu.ee/~biotagene/LIFE_eng/Home.html
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4. Assessment of the environment according to the monitoring results
The BIOTAGENE method allows to assess the status of the environment by
measuring the anthropogenic pressure applied upon it. Correspondingly the baseline
for the method development had to be defined, which in our case was chosen to be the
untouched environment of the primeval forest. The samples collected from the
Järvselja forest protection area in Estonia served as the example of untouched nature.
The mentioned forest has been under protection as a nature reserve since 1924. When
the reserve was established it was considered in those days that the forest had stayed
untouched prior to the date for at leas an other 100 years. The other extreme in our
sample set is represented by samples from oil shale mining spoil tips in eastern
Estonia representing the situation with extreme pressure of human activities upon
ecosystem. In principle all the other 400+ sample collection areas from the countries
around the Baltic Sea can be characterized as falling between these two extremes. The
method has been developed as a first attempt to introduce an objective approach for
environmental monitoring not depending so much as the current approaches upon the
qualification of the monitoring personnel and their subjective prior experience. At the
same time the bases of the method are not so much different from the classical
naturalist approaches of determination and counting of species in the environment,
since in principle the metagenomic approach of exploiting molecular biological
methodology of the BIOTAGENE method performs the same tasks only that the
species determination and counting procedure results are generated independent of the
observer’s subjective prior expertise.
The developed scale for the Pscore works is in general in accordance with the
environment assessment scale in use at the University of Tartu Frontiers in
Biodiversity Research (FIBIR) Centre of Excellence (see Table 2). As an example of
discrepancy between the two approaches is a case of comparing a reindeer pen with
the surrounding tundra in the Skallovaara area in northern Finland. The reindeer pen
got 1 according to the FIBIR scale and the surrounding tundra got 4. The calculated
corresponding Pscore-s gave average of 0.79 for the pen and 0.86 for the tundra while
single samples scored between 0.61 and 0.97 in the first case and 0.79 and 0.96 in the
second one. Correspondingly the BIOTAGENE method classified both sample sets as
ones from ecosystems with limited anthropogenic pressure although the reindeer pen
gave an impression of a very disturbed environment. In this context we see the value
of the Pscore as a significant objective tool in environment assessment assisting the
observer with an extra parameter of the ecosystem’s microbiome status.
The BIOTAGENE method yet being in its infancy of usage for assessment of
environmental status understandably is not meant to replace the current approaches
overnight, but more likely is an objective extra measure while evaluating
anthropogenic pressure. The current version of the method represents a developed
and verified prototype based on the samples collected from 409 defined sites and its
usefulness for universal monitoring has yet to be seen. In the After-Life period the
method has to be evaluated on a wider set of samples from even more varying
environments and the improvement and optimization of the method has to go hand in
hand with the established and classical methodologies, thus providing a thoroughly
verified and accepted measure for monitoring the influence of human activities upon
the environment we live in.
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5. Appendices
APPENDIX I
Manual 1
Collection of soil samples for metageneomic analysis of
microbiome diversity.
Appliances













GPS device
Manual soil drill (for 30 cm depth)
Means for soil drill cleaning (water, chlorine containing detergent 10x
solution, cleaning brush, ladle, 2x 5-10 liter buckets)
Rubber gloves
Disposable plastic tablespoons
Sample collection containers (plastic zipper bags 10x15 cm, Minigrip),
labeled beforehand (date, sample collection site coordinates if needed)
Silica gel (dry, preferably containing humidity marker)
In the case of need the cadastre (land property) or field maps.
The soil sample order form if needed (available at
http://pmk.agri.ee/index.php?valik=150&keel=1&template=template2t
eenused.html)
Accompanying note for the DNA analysis of the soil
samples
Writing implements (pens, pencils)
Measuring instruments (tapeline and ruler)
Paper napkins
Soil sample collection
The sample collection area is selected based on randomness principle inside the
borders of a representative ecosystem (field, grassland, meadow, forest, etc.). On the
selected sample collection area (30x30 m square) the samples are collected from 9
spots on 10 m distance from each other (look scheme). The samples are collected a
straight transect 10 meters apart from each other. Attention should be paid that the
soil at different collection spots should be of the same type and having a similar water
regime. The samples are collected by drilling to the depth of 10-15 cm.
During the sample collection and handling a caution of carrying rubber gloves should
be taken as to minimize the chances of contaminating the samples with human genetic
material. For every new sample a new pair of rubber gloves should be used. The
sample is collected with a manual soil drill prior washed with the detergent and rinsed
clean with clean water using the ladle above the bucket containing the cleaning agent.
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Before collecting the sample the drill is inserted into the soil in full length next to the
sample collection spots, as to clean it from the possible detergent leftovers an as to
balance it with the surrounding soil environment. Before taking the sample the drill is
cleaned of the attached soil without washing.
Next the sample is collected as follows:
 inserted the drill 10-15 cm into soil
 turn the inserted drill 90o
 pull the drill out of soil
 using the disposable plastic spoon empty the soil in drill into the sample
collection container (plastic zipper bags 10x15 cm, Minigrip, containing silica
gel, labeled beforehand)
 close the sample collection container (try to keep the amount of air inside the
container minimal, as to save space later on)
During the sample collection one should pay attention to avoid stony sites. It can
damage the drill. One should also pay attention as to avoid capturing substrates like
dry grass, pieces of manure, small stones or fertilizer granules, the contest of which
could alter the later analysis results.
After moving to the next sample collection spot one should repeat the abovedescribed actions starting with the washing of the soil drill. New rubber gloves and
disposable plastic tools are to be used for collecting every new sample. If the soil
collected occurs to be too humid fresh silica gel should be added to the sample
container (could be also done after returning from sample collection but as soon as
possible).
If the climate allows samples may be collected the all year round, but not in a rainy
weather and the soil should be try enough not to attach to boots hands or the sample
collection appliances.
During the field works also the sample accompanying not should be fulfilled. Every
single sample must be coded and the code should be included in the accompanying
note and on the label of the sample collection container (it simplifies sample
collection if the coding is carried out beforehand). During the sample collection it is
paramount that the following metadata are documented in the accompanying note:
location – it is suggested to use GPS coordinates, time of sample collection, weather
description, description of vegetation and if present the trees, if possible the
description of fauna or the traces of animal activity, if possible photos of landscape in
4 directions and detailed photos of the sample collection spot, the name of the sample
collector, date. In addition the ecosystem of the sample collection site should be
described, including the climate zone and the biogeographic zone of the sample
collection area.
Pay attention to the correctness of sample collection and avoid cross contamination
between the single probes.
NB! No samples should be collected:
 during heavy rain and not before 3-4 hours after the end of rain. Soil should
not be too wet and greasy
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
from places where manure, compost, lime, fertilizers, potatoes, straw/litter,
hay, etc. has been deposited temporarily or for a longer period, unless the
analysis of that kind of samples is the aim of the study
 form spots differing significantly from the general sample collection area, like
micro hollows, plowing furrows where the soil has been agglomerated or
separated due to the successive passes, where layer of soil under the humus
horizon has been ploughed up, from spots on the sample collection area where
small patches of different soil types occur
 from above the drains, from edges of ditches, waysides, close to the water
reservoirs, lakes, rivers, waterholes and places differing significantly from the
general background, unless it not the objective.
NB! The accompanying note should be added to the samples.
This manual has been developed in the framework of the Life+ program
BIOTAGENE project (LIFE08 ENV/EE/000258)
APPENDIX II
Manual 2
Storage of soil samples over a longer period
For extended storage the soil samples should be as dry as possible. That is important
as in dry environment all the life processes are kept minimal and less changes in the
DNA fingerprint of the sample are to be expected.
After bringing samples to the lab it is important latest the next day to examine all the
samples collected during the fieldwork one by one to ascertain that the soil in samples
is dry, the container intact and properly sealed. In the case of a damaged sample
container the sample should be transferred into an intact one marked with a
corresponding label. If the soil sample occurs insufficiently dry silica gel should be
added to the sample container to the amount ensuring the preservation of the sample
in dry conditions over extended periods. As mentioned in Manual 1 (Collection of soil
samples for metageneomic analysis of microbiome diversity) this activity could be
simplified if the silica gel added to sample containers contains the humidity color
indicator. In the case the indicator is showing saturation it is important to add silica
gel to the sample container. In the case that silica gel used lacks the indicator one
should trust one’s own subjective judgment and experience.
It is suggested to pack the sample containers into suitable cartoon boxes marked
accordingly for convenient archiving. In our case we have found most convenient to
use unmarked wine bottle boxes (internal measures 9x9x38 cm) as their sizes fit the
sample containers most adequately.
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LIFE08 ENV/EE/000358
The soil archive should be kept in a dark and dry environment, if possible in a room
with a controlled temperature.
In our experience no significant alteration in the microbiome characteristics occur in
the soil samples stored the way described over a period of least two and a half years.
This manual has been developed in the framework of the Life+ program
BIOTAGENE project (LIFE08 ENV/EE/000258)
APPENDIX III
Manual 3
Preparation of soil samples for DNA purification
The preparation of soil samples for DNA purification essentially means carrying out
the procedure of weighing the required amount of soil sample into Eppendorf 2.0 ml
Safe-Lock micro test tubes containing grinding material, which we further refer to as
“homogenization tubes”. Alternatively one can use 2.0 ml screw cap v-bottom tubes
(it is suggested to use in this case the caps with an attachment loop to avoid the-mix
up of the caps during handling) to avoid the possible spillage of the samples during
handling.
To complete the preparation of homogenization tubes 0.5 g of 0.4-0.6 mm in diameter
of ZIRPRO beads should be weighed into every tube. (Saint-Gobain, #ER 120S
http://www.zirpro.com/uploadedFiles/SGzirpro/Documents/SGZirPro-ER120-TDS201204-E.pdf). The weighing procedure must be carried out on clean surfaces, in a
clean lab coat and carrying clean powder free rubber/latex gloves, a mask and
covering ones hair to minimize the contamination of the samples with human
originated genetic material.
As an alternative one can use Mo Bio Laboratories Inc. 0.7 mm Garnet Bead Tubes
(www.mobio.com; cat# 13123-50) already containing desired grinding material.
To start the procedure one should find in the archive the required for the study soil
samples and prepare the corresponding amount of 2.0 ml homogenization tubes by
labeling them with the sample names or numbers or any convenient codes, as it is
usually very unlikely the whole sample label will fit on the tube. To avoid later mixup of the samples between each other during the downstream steps of the DNA
purification and PCR sample preparation it is strongly suggested to create at this stage
a conversion table with the corresponding sample labels adjacent to the sample codes
on the homogenization tubes.
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LIFE08 ENV/EE/000358
Before starting the transfer of the weighed samples into the homogenization tubes one
should thoroughly verify that the sample label code conversion table and the codes on
the tubes correctly correspond to each other. It will avoid any unnecessary confusion
and disturbance during the sample weighing activities.
While choosing a room or area for sample weighing one should keep in mind that the
dry soil samples are prone to great quite some dust and thus cause cross
contamination between samples being processed as well as with the ones to be
processed in the future. Correspondingly before initiating the activity one should
clean thoroughly all the surfaces to be used including the balances. To avoid or
minimize the contamination of the whole room it is suggested to perform the sample
weighing procedure in the chemical hood or under a local hood above the weighing
area. Such a precaution will minimize the possibility of cross contamination, as the
soil dust created will be sucked out of the room.
To minimize the possible contamination during weighing the samples with DNA
originating from humans other than the material from the field (from hair, skin, saliva,
etc) it is suggested to carry a clean lab coat, powder-free rubber or latex gloves, a
mask covering ones nose and mouth and a cap covering ones hair. In the case the
gloves come in contact with the soil from the sample during opening/closing and
handling of the sample container one should put on a new pair of cloves prior to
starting with the next sample.
For sample weighing it is suggested to use digital balances, which display at least
tree positions after coma (0.000 g), an instrument with mg-accuracy presumably.
Weighing the soil directly into the homogenization tubes might be inconvenient due
to the small diameter of the tube’s orifice resulting in spillage of the soil on the
balance surface and thus creating threat of cross contamination. It is suggested to use
the smallest available disposable weighing boats. 250-300 mg of soil from every
sample should be weighed on the disposable weighing boat by using a disposable
plastic teaspoon. It might be more convenient to use the tip of the spoon’s handle
rather than the spoon itself for transferring the soil. In next step the weighed soil
should be carefully transferred into the homogenization tube and the tube closed. The
used weighing boat and the spoon used for weighing should be disposed and new
clean ones taken for the next sample.
In the described way the samples can be prepared in advance and kept for a time
being before proceeding with the DNA purification. In the case of a longer period it is
suggested to keep all the samples for the planned experiment in a larger intact
Minigrip bag and add some silica gel to control the possible humidity levels in the
prepared samples.
This manual has been developed in the framework of the Life+ program
BIOTAGENE project (LIFE08 ENV/EE/000258)
15
LIFE08 ENV/EE/000358
APPENDIX IV
Manual 4
Purification of total DNA from soil samples
The first step starting the DNA purification from the soil samples is to choose the
prepared (according to Manual 3: Preparation of soil samples for DNA purification
for sequencing) homogenization tubes with desired soil samples. Prior to starting the
DNA purification prepare two sets (one for DNA binding and one for the collection
and preservation of the purified DNA) of 1.5 or 2 ml safe-lock or screw-cap tubes
equivalent to the amount of samples in the experiment by labeling them with the
corresponding sample codes.
Further on is given a step-by-step protocol for soil DNA purification.
1) Add 800 l of Lysis buffer to every tube. (In the case of a sediment present in
the lysis buffer heat it to 60oC for 10 min prior to usage)
2) Vortex to mix evenly the homogenization beads with the soil in the sample
and the lysis buffer
3) Attach the Mo BioVortex Adapter for Vortex-Genie® 2 to a Vortex Genie® 2
Vortex. Insert the homogenization tubes into the adapter (if there are less than
24 tubes in the procedure pay attention to achieve a balanced distribution of
tubes in the adapter)
4) To homogenize the samples switch the vortex on for 20 min at max speed
5) After homogenization centrifuge the tubes for 2 min at 104 g
6) Transfer 300 l of supernatant to the first set of pre-prepared tubes. Save the
leftover for backup
7) Add 20 l SiMAG/MP-DNA beads to the transferred supernatant. (Before
transferring the beads mix the stock thoroughly by vortexing)
8) Add 300 l of binding solution and mix on the Vortex
9) Attach the tubes to the Mo BioVortex Adapters for Vortex-Genie® 2 and mix
for 5 min at max speed
10) Insert the tubes into the magnetic stand and allow the bead separation for 1-2
min
11) Aspirate carefully as much of the liquid from the tubes as possible using a
pipette leaving the magnetic beads behind
12) Remove the tubes with beads from the stand and add 500 l of salt wash
solution to every tube. Mix
13) Insert the tubes into the magnetic stand and repeat the actions from step 10)
to12) twice, resulting in three washes of the beads with the salt wash solution.
Special attention should be paid during the last aspiration of the salt solution
to get rid of the solution as completely as possible.
14) Take tubes out of the magnetic stand and add 500 l of ethanol wash solution
to the tubes. Mix
15) Insert the tubes into the magnetic stand and allow the bead separation for 1-2
min
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LIFE08 ENV/EE/000358
16) Aspirate the ethanol wash solution by leaving the beads behind.
17) Repeat the steps from 14) to 16)
18) After removing the final ethanol wash solution leave the open tubes with
beads to dry for ~5 min at room temperature until the ethanol has evaporated
19) Add 100 l of elution buffer to the tubes and pay attention that all the beads
end up in the solution.
20) Incubate the beads in the elution buffer for minimum 1-2 min. No need to
exceed 5 min.
21) Remix the beads with the elution buffer and insert the tubes into the magnetic
stand for 1-2 min
22) Transfer the elution buffer containing the DNA to the second set of preprepared tubes.
23) Dispose of the tubes with the leftover of beads and store the tubes with the
purified DNA for further use at -20oC.
Solutions
Lysis buffer for 96 samples (85 ml):
Chemical
Stock
concentration
Tris-HCl (pH 8.0)
1M
EDTA (pH (8.0)
0.5M
NaCl
5M
SDS
100%
Rnase A
100 mg/ml
H2O (MilliQ)
Binding solution for 96 samples (40 ml)
Chemical
Stock
concentration
PEG-8000
100%
NaCl
5M
H2O (MilliQ)
Volume to add
8.5 ml
17 ml
17 ml
1.7 ml
85 l
up to 85 ml
Amount to add
8.0 g
32 ml
up to 40 ml
Final
concentration
100 mM
100 mM
1M
2%
100g/ml
Final
concentration
20%
4M
Salt wash solution for 3x96 samples (150 ml)
Chemical
Amount to add
GITC
70.75 g
H2O (MilliQ)
up to 150 ml
Ethanol wash solution for 2x96 samples (106 ml)
Chemical
Stock
Volume to add
Final
17
LIFE08 ENV/EE/000358
Ethanol
H2O (MilliQ)
concentration
96%
77,3 ml
28.7 ml
Elution buffer for 96 samples (11 ml)
Chemical
Stock
Volume to add
concentration
Tris-EDTA (TE) 1.0M Tris Base and 0.11 ml
100x
0.1M EDTA
H2O (MilliQ)
10.89 ml
concentration
70%
Final
concentration
1x
Chemicals and appliances:
Vortex Genie® 2 Vortex
Mo BioVortex Adapters for Vortex-Genie® 2 cat# 13000-V1-24
SiMAG/MP-DNA beads
Magnetic stands (choose the desired model corresponding to your needs by
following the given links): Promega, Life Technologies, Millipore
This manual has been developed in the framework of the Life+ program
BIOTAGENE project (LIFE08 ENV/EE/000258)
APPENDIX V
Tabel1. Primer sets for the BIOTAGENE method
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
start
523
len tm
gc% oligo seq
20 59.96 50
GTTCGGATTTACTGGGCGTA
769
20
60.03 50
CGGGGTATCTAATCCCGTTT
618
20
60.01 60
GTGCTAGAGTGCAGAAGGGG
1013 20
59.88 50
TACAGGTGGTGCATGGTTGT
1255 20
60.07 55
TGGGATTAGCTCCACCTCAC
1036 20
60.05 55
CAGCTCGTGTCGTGAGATGT
263
20
59.96 50
TAATGGCCTACCAAGGCAAC
426
20
60.01 45
CCGAAAATCTTCATCCTCCA
283
20
60.18 55
GATCAGTATCCGGCCTGAGA
Sequence name
gi|2329870|emb|Z95709.1|
Bacterial species 16S rRNA
gene (clone 11-25);
INDICATOR 1
gi|311335259|gb|HQ327138.1|
Arthrobacter sp. TP-Snow-C29
16S ribosomal RNA gene,
partial sequence
INDICATOR 2
gi|192806377|emb|FM176885.1|
Uncultured Acidobacteriaceae
bacterium partial 16S rRNA
gene, clone CL6-7.L265
INDICATOR 3
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LIFE08 ENV/EE/000358
OLIGO
LEFT
138
PRIMER
RIGHT
347
PRIMER
HYB
167
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
20
59.93 50
TGGGATAACAGTCCGAAAGG
gi|110754887|gb|DQ829574.1|
Uncultured Chloroflexi
bacterium clone
DOK_NOFERT_clone663 16S
ribosomal RNA gene, partial
sequence
INDICATOR 4
gi|309241479|emb|FN659097.1|
Uncultured bacterium partial
16S rRNA gene, clone
t0_12L41
INDICATOR 5
20
60.11 60
CCACCCGTAGGTGTATGGAC
20
60.08 45
ACCGCATGTGGTTATTGGAT
483
20
60.1
55
TCCAGACGGTACCTCCAAAG
664
20
60
55
GCCTCAAGTCCTGCAGTTTC
617
20
60.02 50
CGTACGGCTCAACCGTATTT
489
20
59.96 50
GTTCGGAATTACTGGGCGTA
661
20
59.97 50
CCACTGGTGTTCTTCCGAAT
556
20
60.24 50
CAACTCCGGAACTGCCTTTA
878
20
59.99 50
GCAAGGCTGAAACTCAAAGG
1116 20
59.97 45
ATGATGGCAACACAGGACAA
1048 20
60.05 55
CAGCTCGTGTCGTGAGATGT
992
20
60.02 60
1153 20
59.93 50
1070 20
60.05 55
GCTAGACAACGGAGGACAGC gi|134021165|gb|EF019741.1|
Uncultured bacterium clone
GAGTGCCCACCTGAAATGAT Elev_16S_915 16S ribosomal
RNA gene, partial sequence
CAGCTCGTGTCGTGAGATGT INDICATOR 8
290
20
59.83 60
CTGAGATACGGCCCAGACTC
515
20
59.96 50
TACGCCCAGTAATTCCGAAC
383
20
60
50
AGGCCTTCGGGTTGTAAAGT
155
20
60.27 50
GATAAGCCCTTACGGGGAAA
389
20
59.96 55
ACCCTAGGGCCTTCATCACT
233
20
60.28 55
ACCAAGGCGACGATCAGTAG
117
20
59.95 55
CTGCCCTGTGGTAGGGAATA
312
20
59.83 60
GAGTCTGGGCCGTATCTCAG
gi|307940623|gb|HQ114170.1|
Uncultured bacterium clone
V201-155 16S ribosomal RNA
gene, partial sequence
INDICATOR 6
gi|134021453|gb|EF020029.1|
Uncultured bacterium clone
Elev_16S_1424 16S ribosomal
RNA gene, partial sequence
INDICATOR 7
gi|146429810|gb|EF220626.1|
Uncultured alpha
proteobacterium clone FI1M_G07 16S gene, partial
sequence
INDICATOR 9
gi|254933897|gb|GQ342579.1|
Bradyrhizobium sp. RI270 16S
ribosomal RNA gene, partial
sequence
INDICATOR 10
gi|110754526|gb|DQ829213.1|
Uncultured proteobacterium
clone
DOK_NOFERT_clone239 16S
19
LIFE08 ENV/EE/000358
HYB
OLIGO
165
20
59.92 50
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
406
20
59.78 50
622
20
59.91 55
516
20
60.03 55
1007 20
60.05 55
1177 20
60
1136 20
60.59 50
77
20
59.73 60
248
20
60.28 55
119
20
60.2
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
LEFT
PRIMER
RIGHT
PRIMER
HYB
OLIGO
78
20
59.97 50
304
20
59.95 60
165
20
59.98 55
783
20
60
978
20
59.99 55
928
20
60.43 45
55
50
50
TGAGTCCTTCGGGAGAAAGA
ribosomal RNA gene, partial
sequence
INDICATOR 11
CGGGGAAGATAATGACGGTA gi|148249196|gb|EF605874.1|
Unidentified bacterium clone
17_L1_BULK_SP6 16S
ACTCGCAGTTCCACTCACCT
ribosomal RNA gene, partial
CACGTAGGCGGCTTCTTAAG sequence
INDICATOR 12
CAGCTCGTGTCGTGAGATGT gi|134021722|gb|EF020298.1|
Uncultured Acetobacteraceae
ATGACGTGTGAAGCCCTACC bacterium clone
Elev_16S_1845 16S ribosomal
RNA gene, partial sequence
TCAAGTCCTCATGGCCCTTA
INDICATOR 13
GGCAGACGGGAGAGTAACAC gi|110754277|gb|DQ828964.1|
Uncultured proteobacterium
clone
CTACTGATCGTCGCCTTGGT
DOK_CONFYM_clone756 16S
GGAACAACCCAGGGAAACTT ribosomal RNA gene, partial
sequence
INDICATOR 14
TGCTAGATTGATGGCGAGTG gi|311335124|gb|HQ318603.1|
Uncultured soil bacterium clone
DLQAMB9C100 16S
GCTGATCGTCCTCTCAGACC
ribosomal RNA gene, partial
ATACCGCATACGACCTGAGG sequence
INDICATOR 15
ACGCCCTAAACGATGTCAAC gi|298397684|gb|HM112378.1|
Uncultured beta
AGTGGCATGTCAAGGGTAGG proteobacterium clone
SHOB681 16S ribosomal RNA
GGATTAATTCGATGCAACGC gene, partial sequence
INDICATOR 16
This manual has been developed in the framework of the Life+ program
BIOTAGENE project (LIFE08 ENV/EE/000258)
20