Alberts Lab Fecal Protocol, 30 Oct 2012
ALBERTS LABORATORY PROCEDURES FOR FECAL
GENOTYPING AND PATERNITY ANALYSES
Prepared by Jacob Gordon, with significant inspiration from the Altmann Lab Protocol
Alberts Lab Fecal Protocol, 30 Oct 2012
FECAL SAMPLE COLLECTION
Samples are collected ad libidum from free-living baboons living in the Amboseli
basin of Kenya. When a known animal is observed defecating, we collect the freshlydropped sample within minutes of its deposit. Unlike the samples collected for
hormone analysis, the fecal sample is not homogenized prior to collection. Instead, a
small portion is removed from the leading edge of the fecal bolus (if possible) and
placed in a plastic 20-mL vial (Evergreen Scientific 220-3519- 080; caps 300-3532G20) prefilled with 95% ethanol (EtOH). The sample if broken up to saturate it with
EtOH and the vial is identified with the baboon’s name (ID), the sample date, and
time of collection. Samples are stored in an evaporatively cooled hut (a “charcoal
fridge”), which keeps temperatures 15C-25C (~daily min, max). A list of all
samples, including the baboon id, the date and time of collection, and any additional
notes about the sample, accompanies the samples when they are eventually sent to
Nairobi.
Unlike the hormone fecal samples, the genetics samples do not undergo any
processing in Nairobi. They are stored there and eventually shipped to Duke
University.
RECORDING IN BABASE, STORAGE
Upon arrival at Duke, the shipped feces are sorted and inventoried by at least one
technician (ideally two, to screen for typos). Tubes are stored in freezers at -85C.
A record of each sample, including its name, collection date, time of collection, and
location in the freezers, is maintained in our online database, Babase. The database
also assigns a unique Tissue ID number (TID), which is referred to below.
DNA EXTRACTION
Until ~late 2009, fecal DNA extractions were performed using the Qiagen DNA Stool
Mini Kit (cat. #51504). Since then, extractions have been done with a protocol from
Qiagen using the Qiagen QIAxtractor, modified somewhat to accommodate the
peculiarities of fecal DNA extraction. That protocol is outlined below:
 Preparation
o Turn on 70°C water bath (not necessary if you plan to stop at end of fecal
transfer)
o For cleaning spatulas (more info below), set up 4 50-mL falcon tubes:
 Two with 40-45 mL dH2O
 One with 40-45 mL bleach
 One with 40-45 mL 95% EtOH
 Line up tubes next to each other in the following order: water,
bleach, water, EtOH
o In a biosafety cabinet, ignite a Bunsen burner, EtOH burner, or other open
flame
Alberts Lab Fecal Protocol, 30 Oct 2012


o Check the bottle of ASL buffer for precipitation. If precipitate is present,
place in the heating-up water bath and allow the heat to dissolve the
precipitate. (not necessary if you plan to stop at end of fecal transfer)
o Don a gown, hairnet, and facemask
Fecal Transfer
o Using metal spatulas, transfer fecal samples into 2-mL round-bottom
centrifuge tubes up to the 0.5 mL mark.
 If it's possible to identify the frontmost part of the fecal bolus, try
to collect from there. We expect a higher amount of baboon
epithelial cells there.
 Be careful to not go above 0.5 ml; if anything, err on the side of
caution and go less.
 We prefer 16 samples per QIAxtractor run
 Use a clean spatula for each sample
 To clean spatulas (be sure to do this with spatulas at beginning of
procedure, before doing any transfers) :
 After using a spatula, place it in first water tube
 When 4-6 spatulas accumulate in first tube, move them to
the bleach tube and allow them to soak for at least a few
seconds. It's okay to continue transfers for 4-6 more
samples and move spatulas out of bleach when the next
group is ready to go in.
 Remove from bleach soak and move spatulas to the second
water tube to rinse bleach. Extended soak is not necessary,
just be sure rinse does occur.
 Move spatulas from second water tube into EtOH.
Extended soak not necessary.
 After EtOH tube, wave EtOH-soaked spatulas over open
flame and allow ignited EtOH to clean and dry spatulas.
Don't get burned!
 Lay spatulas across a lab marker or some way such that the
ends that will be touching feces aren't touching anything.
 Water tubes should be emptied and refilled after each
extraction, but bleach and EtOH tubes can usually be reused
for 2-3 total extractions. When in doubt, throw it out.
o If necessary, it's okay to pause the procedure at this point
Lysis
o If you paused the procedure at fecal transfer and are resuming now, turn
on 70°C water bath, and if precipitation in ASL buffer has occurred, heat it
up as described above
o To each tube, add ASL buffer (make sure precipitate is not present in
buffer first) up to near the top, ~1600 µl.
 This feces:buffer ratio is fairly sensitive. As mentioned above, it's
better to err on the side of having less feces; DNA yield decreases
Alberts Lab Fecal Protocol, 30 Oct 2012

sharply as the amount of feces increases, presumably due to
inhibitors in the feces.
 It's important to not stop at this point. If precipitate from ASL
forms again, DNA will likely precipitate with it and may not return
to solution if heat is re-applied. We have heard from collaborators
that overnight incubation at 37°C (which should be sufficient to
prevent precipitation) increases fecal DNA yield in other kits, but
we haven't tried it enough with the QIAxtractor to know.
o Make sure all caps are securely and firmly closed, and vortex all tubes for
at least 15 minutes, or until all samples are homogenized.
o Incubate homogenized samples in 70°C water bath for at least 5 minutes
Centrifugation/Supernatant Collection
o Centrifuge tubes at maximum speed (~14000 RPM, ~18000 × g) for 10
minutes
 Spinning less time than this tends to not sufficiently pull down
invisible contaminants that often cause problems later on in the
QIAxtractor
 Spinning more time than this may (we haven't done any
experimentation on this question) pull some DNA into the pellet
o Transfer supernatant to separate wells of a QIAxtractor lysis plate, 200 ul
supernatant/well. When doing 16 samples, we use the following layout:
Samp
1
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Samp
3
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Take extreme care to avoid any insoluble matter when
transferring
 Fecal lysates often have a mucusy material that usually
rests at the top of the pellet; this matter often causes
membrane clogs in the QIAxtractor
 There are other particulates, often invisible at this stage,
that can cause clogs in QIAxtractor, so keep pipet tip as far
from pellet as possible
QIAxtractor
o Bring plate to the QIAxtractor
o For DNA capture, we find that the VX capture plate (Qiagen cat. #950921)
gives much, much better yields of endogenous DNA than the DX plate
(#950911).
Alberts Lab Fecal Protocol, 30 Oct 2012
Aside from the VX capture plate, use only DX reagents (they're the
same price or cheaper)
 We've tried using all VX reagents, but saw no notable
improvement in DNA yield.
o Reagent Prep
 DXW, DXF, and DXE (we use autoclaved ddH2O instead of DXE) can
be poured into respective reagent troughs far in advance, like
during sample vortexing, heating, or centrifugation. For each
trough, add the volume directed by the software wizard.
 DXB binding additive (refrigerated white solid) needs to be added
to buffer DXB, but once added they only stay stable for a month at
most. If we expect to do a lot of extractions, we mix one vial of
binding additive with one bottle of DXB and use the quantity that
the software's wizard directs. If we expect few extractions in the
near future, the QIAxtractor manual has an appendix showing
precise amounts (in mg) of additive to add to desired amount of
DXB, and we mix only the amount we need
 Binding additive needs to be refrigerated when solid, but
once added to buffer, do not refrigerate buffer DXB. Cold
storage causes precipitation of the binding agent, so store
at room temperature.
 Mixed DXB is photosensitive, so don't add DXB until
just before DXL and before starting cycle
 DXL: dilute digest enzyme (DX) 1:10 in DXL buffer to a final
volume of whatever the software's wizard tells you it needs.
 Once diluted, the enzyme doesn't stay stable for long,
so do this just before starting the cycle
o Dealing with clogs
 There are two steps in the QIAxtractor protocol where the
program waits for confirmation from the user before it will
proceed onward. Before allowing the procedure to continue, it's
important to look at the wells and make sure no wells are
clogged—they should be dry. If fluid is still present in any wells,
open the lid and try pipetting the residual liquid up and down a
few times to dislodge whatever clog is present. If the clog is
dislodged, you'll see the movement of fluid pretty soon. If no
movement occurs, manually aspirate fluid as best you can without
puncturing the membrane, and then allow the procedure to move
onward.
 If any clogs are visible at the second checkpoint, check on the plate
periodically for the rest of the cycle to make sure reagent added to
the clogged well(s) doesn't overflow into adjacent wells.
After QIAxtractor
o Elution tubes from QIAxtractor are not good for long-term storage. No
matter how tightly you place the caps on, cold storage causes the caps to


Alberts Lab Fecal Protocol, 30 Oct 2012
pop and the fluid sublimates over time in the freezer. Storage for a night
or two isn't likely to be harmful, but it's best to transfer eluted DNA's to
screwcap tubes as soon as possible.
Each extracted DNA sample is assigned a DNA ID number (DID), and we record basic
information about the sample, including name, extraction date, technician(s)
involved with extraction, and the TID of the tissue source. All this information is
stored in Babase.
After extraction and transfer to screwcap tubes, DNA's are stored at -20C.
qPCR QUANTIFICATION
Fecal DNA extracts are expected to have large amounts of non-baboon DNA
(especially bacterial), so spectrophotometric quantification (e.g. Nanodrop) is not
very helpful. Instead, we quantify our fecal DNA extracts using a qPCR assay for
mammalian c-myc, described in Morin et al, Molecular Ecology 2001. We order
probe and primers from IDT, and use the Qiagen Quantifast Probe PCR Kit (cat.
#204254), with the following recipe, from the kit's manual:
Water
2X Mastermix
10M Forward Primer
10M Reverse Primer
10M Probe
1X
8 l
12.5 l
1.0 l
1.0 l
0.5l
Template
Total Volume
2.0l
25l
50X
400 l
625 l
50 l
50 l
25 l
100X
800 l
1250 l
100 l
100 l
50 l
PCR conditions:
1  95C, 10 mins
40  95C for 15 sec and 59C for 30 sec
For our standard, we use ABI's TaqMan Control Genomic DNA (cat. #4312660),
serially diluted 1:4, from 10 ng/L (stock) down to 9.8 pg/L.
Concentration according to qPCR is recorded in Babase, and should be imported to
Babase as soon as possible after qPCR is complete.
MICROSATELLITE PCR
To determine an individual's genotype, we PCR at 14 distinct microsatellite loci,
using Qiagen's Multiplex PCR Kit (cat. # 206143, 206145). Qiagen's protocol is for
50-L reactions; we scale down to 10 L, as shown below:
Water
1X
To 10 L
Alberts Lab Fecal Protocol, 30 Oct 2012
5 L
1 L
1-2 L
2X Mastermix
2 M Primer Mix*
DNA
*Primer Mix is a mixture of forward and reverse primers for all loci being assayed in
the reaction. Each individual primer has a 2 M concentration, so that the final
concentration in the PCR of each primer will be 0.2 M.
Primer Info:
Locus
Size
Range
DNA/well
(L)
Fwd Primer
Rev Primer
AGAT006
131-181
1
AGTGGATCGATAGATTGACAGATG
TCAGGTGACAGCCAAGTCAATTCA
D10s611
150-195
1
CATACAGGAAACTGTGTAGTGC
CTGTATTTATGTGTGTGGATGG
D11s2002
252-280
2
CATGGCCCTTCTTTTCATAG
AATGAGGTCTTACTTTGTTGCC
D13s159B
165-181
2
ACACCTCTCCCAGTTGTTGG
CAACTCCAGGCCAAATCATC
D14s306
146-190
1
AAAGCTACATCCAAATTAGGTAGG
TGACAAAGAAACTAAAATGTCCC
D18s851
225-249
2
CTGTCCTCTAGGCTCATTTAGC
TTATGAAGCAGTGATGCCAA
D1s1656
130-167
1
GTGTTGCTCAAGGGTCAACT
GAGAAATAGAATCACTAGGGAACC
D2s1326
237-273
>=2
AGACAGTCAAGAATAACTGCCC
CTGTGGCTCAAAAGCTGAAT
D3s1768
178-218
>=1
GGTTGCTGCCAAAGATTAGA
CACTGTGATTTGCTGTTGGA
D4s243
155-179
1
TCAGTCTCTCTTTCTCCTTGCA
TAGGAGCCTGTGGTCCTGTT
D5s1457
110-138
1
TAGGTTCTGGGCATGTCTGT
TGCTTGGCACACTTCAGG
D6s501
171-227
2
CTGGAAACTGATAAGGGCT
GCCACCCTGGCTAAGTTACT
D7s503
133-169
2
ACTTGGAGTAATGGGAGCAG
GTCCCTGAAAACCTTTAATCAG
D8s1106
128-161
1
TTGTTTACCCCTGCATCACT
TTCTCAGAATTGCTCATAGTGC
Using IDT and ABI to generate our oligos, we use the above primer pairs, with one of
the primers labeled with the dyes FAM, HEX, or NED. Each PCR well usually uses
primer multiplexes of 3-4 pairs. In designing multiplexes, we take care to not use
the same dye for oligos of overlapping size range.
Cycle Conditions:





1  10 minutes, 95C
16 
o 15 seconds, 95C
o 30 seconds, 66C (-1C each cycle)
o 30 seconds, 72C
29 
o 15 seconds, 95C
o 30 seconds, 50C
o 30 seconds, 72C
1  3 minutes, 72C
Hold at 10C
Alberts Lab Fecal Protocol, 30 Oct 2012
After PCR, we store plates at -20C if necessary, but ideally we submit the 96-well
plates to Duke's IGSP Sequencing Facility as soon as possible for fragment analysis.
Information about each locus (not reaction) that we PCR, including locus name, DID,
and reaction date, is recorded in Babase using a unique reaction ID (RID) for each
locus.
For example, the PCR in a single well may use a primer multiplex that will
amplify four distinct loci, so four distinct RID's will be recorded for that one
well.
PCR FRAGMENT ANALYSIS
Finished PCR reactions are submitted to Duke's IGSP Sequencing Facility, where
they're diluted and analyzed by an ABI 3730xl DNA Analyzer.
Data are returned to us in .fsa files, one for each PCR well. Using SoftGenetics'
GeneMarker software, we analyze the returned data to call observed alleles at each
locus. We record these observations in Babase, with each allele call associated with
its corresponding RID.
FINALIZING GENOTYPES
Using several replicate PCR's for each individual at each locus, and comparing
observed alleles with genotypes of the individual's mother (when known), we
eventually arrive at a "consensus" genotype for each individual at each locus. These
are regularly recorded in Babase.
PATERNITY ANALYSIS
For paternity analyses, we use Cervus, by Field Genetics. To do this, we submit the
list of finalized genotypes recorded in Babase and a list of each offspring with their
mother (when known) and the names of all the kid's potential fathers. All of this
information is recorded in Babase.
We do replicate analyses, using confidence levels of 80%, 95%, and 99%. We then
record the paternities in Babase, along with the highest confidence level at which
Cervus called that paternity.