The Importance of DNA Extraction
in Metagenomics: The Gatekeeper
to Accurate Results!
ABRF 2013 Research Study
Nucleic Acids Research Group
(NARG)
Preparing the NARG Metagenomics
Bacterial Cocktail
• Bacteria were grown to stationary phase (2 weeks) on
TSA solid media
• One loop-full (2mm) of cell mass was suspended in 10
ml nuclease-free PBS with 30% Ethanol for 72 hours
(to fix), pelleted via centrifugation, washed in PBS and
re-suspended in 0.02% sodium azide/PBS to 5ml.
• Samples were diluted 1:100 and enumerated
microscopically using Sybr Green/Acridine orange
with the C-chip micro- hemocytometer at 650 X
Note: Viable heterotrophic plate counts were not used
because they dramatically underestimate populations
Sample prep continued
• Samples were pooled to create cocktail
• Test extraction were performed to assure
enough yield for NextGen sequencing
(20 to 50 ngs total DNA)
• Shipping tubes were prepared by distributing
80ul of the bacterial cocktail containing 1.1 x
10^8 cells
Note: Assume 4 fg/cell would yield
approximately 430 ng total
Microbial Table
GC
Calculated as
Shipped
Motile Spore forming
38
9.28E+06
Rod
Motile Spore forming
35
4.80E+06
-
Rod
Purple nonsulfur phototrophic
64
9.28E+06
13881
+
Cocci
Spore Forming
42
9.92E+06
Enterococcus faecalis
19433
+
Cocci
Non motile
38
9.92E+06
Pseudomonas aeruginosa
27853
-
Rod
Non-spore forming
67
7.04E+06
Enterobacter aerogenes
13048
-
Rod
Non-spore forming
53
1.22E+07
2228
+
Coccci
Non-spore froming
32
2.46E+07
Klebsiella terrigena
33237
-
Rod
Non-spore forming capsule forming
58
1.02E+07
Micrococcus luteus
4698
+
Cocci
Non-spore forming
72
9.60E+06
10137
+
Filament Mycelia and terminal Spore forming
72
1.31E+06
Microbe
ATCC #
Gram
Bacillus megaterium
14581
+
Rod
Bacillus cereus
11778
+
9791
Sporosarcina ureae
Rhodospirillum rubra
Staphylococcus epidermidis
Streptomyces griseus
Morphology
Cultures were assembled to mathematically achieve 1.1 x108
1.08E+08
Microbe Percent in Synthetic Cocktail
Streptomyces
griseus
1%
Micrococcus
luteus
9%
Klebsiella
terrigena
9%
Bacillus
megaterium
9%
Bacillus cereus
4%
Rhodospirillum
rubra
9%
Sporosarcina
ureae
9%
Staphylococcus
epidermidis
23%
Enterococcus
faecalis
9%
Enterobacter
aerogenes
11%
Pseudomonas
aeruginosa
7%
Microbe Microscopy
Experimental Design
(DNA isolations)
9 different methods selected
7 methods (samples isolated in duplicate)
1 method was performed at two different labs
(duplicate samples), different procedures
modified
per manufactures recommendation
1 method (single sample)
Mo Bio PowerSoil kit
adapted -A
Mo Bio PowerSoil kit-B
REDExtract-N-Amp
Tissue PCR Kit
Transfer sample to 200ul PCR tubes
TWEAK:
Sample added to Bead
Tube then homogenized
on bead beater 30
seconds at 4200RPM
w/Vortex Genie
Add 20ul Extraction buffer (including
0.25 volumes of tissue prep solution)
Incubate RT 10 minutes
Incubate in thermocycler
65C 10 minutes, then
95C 10 minutes
Add 20ul neutralization buffer
Briefly vortex to mix
Transfer into 1.5mL tubes for shipping
(combining by rep)
Modified CTAB
Modified from K. DeAngelis et al.
Environmental Microbiology 12, 31373149 2010, and Jenni Hultman (J.
Jansson lab) protocol
1. To each tube at 500ul CTAB buffer and 50ul
AmAIS, vortex
2. Add 500ul P-C-IAA (in fume hood), vortex
3. Shake 5.5 m/s for 30 seconds
4. Centrifuge 16000xg 5 minutes 4C
5. Prepare new tubes, add 500ul CHCl3
6. Transfer top aqueous layer from tubes to CHCl3
tubes, vortex
7. Centrifuge 16000xg 5 minutes 4C
8. Prepare new tubes, add 1mL PEG 6000
9. Transfer top aqueous layer to PEG tubes, vortex
10. Incubate RT overnight
11. Extract second time from same original lysed
soil sample. Add another 0.5 mL CTAB to the
lysis tube (pellet in step 6) and proceed from
step (1) above. Reuse the same CHCl3 tubes
12. After O/N incubation of second
extraction, centrifuge 16000xg 10+ minutes 4C
13. Pour off PEG 6000 solutions, remove excess
viscous liquid as possible with pipet without
disturbing pellet
14. Wash in 500mLO cold 70% EtOH. Spin 16000xg
5 minutes 4C
15. Dry pellets ~ 5m RT (not totally dry) and
resuspend pellets in total 50ul buffer EB.
epicenter® SoilMaster™
DNA Extraction Kit
1.250µl of Soil DNA Extraction buffer + 2µl of
Proteinase K; vortex briefly.
2. Shake the tube at 37⁰C for 10 minutes.
3. Add 50µl of Soil Lysis Buffer and vortex
briefly.
4. Incubate at 65⁰C for 10 minutes.
5. Add 60µl of Protein Precipitation
Reagent, mix thoroughly by inverting the
tube.
6. Incubate on ice for 8 minutes. Centrifuge
the tube for 12 minutes at maximum speed.
7. Transfer the supernatant to a new 1.5-ml
lo-bind tube.
8. Add 6µl of DNA Precipitation
Solution, vortex briefly. Incubate at room
temperature for 5 minutes.
9. Centrifuge for 5 minutes at maximum
speed. 10.Carefully remove the supernatant.
11. Wash the pellet with 500µl of Pellet
Wash Solution.
12. Invert to mix then spin for 3 minutes at
maximum speed. Carefully remove the
supernatant.
13. Repeat the wash and spin.
14. Resuspend the pellet in 12µl of TE Buffer.
Qiagen Gentra
PureYeast & Bacteria
Kit
Lyticase
Protease
Rnase A
Sample Prep
1. Multi-enzyme digestion 37oC
5 hrs +O/N RT
ReadyLyse – 2400U
Mutanolysin - 7U
•
Achromopeptidase- 1200U
Lysostaphin - 8U
•
Lysozyme - 100ug
Lyticase - 30U
•
Chitinase -100U
Modified Omega Insect
Kit
PrepMan® Ultra Kit
Modified
Add 350 PCI and Vortex
1min
Spin and Xfer supernatant
to new tube
Add equal amount of
Omega CBL and follow SOP
NOTE: This reagent is not designed
for metagenomics. It is intended for
simple “lyse-n-amp” of a single
bacterial culture-
•
2. Boil 5 min
3. Proteinase K digestion
(20mg/ml) 37oC 5hrs +RT O/N)
Modified PrepMan®
Ultra with Phenol and
SDS
• Add 200ul of PrepMan
Ultra
4. Add Ceramic Ball (MP bio) and
100mg of 1mm Diamond:ALO3
• Add 5% SDS
abrasive (200um)
• 5 sec COVARIS (20%10Int-1000b)
• Add 250ul Chloro:IAASpin- Xfer Supernatant
• Add 2x volume ETOH
• Apply to Qiagen Gel
5. FastPrep 4K rpm 30 sec
band extraction column
•
•
•
•
•
•
Add 200ul of PrepMan
Ultra
Add 350ul of PCI
5 sec COVARIS (20%10Int-1000b)
Spin and Xfer
supernatant
Add 250ul Chloro:IAASpin- Xfer Supernatant
Add 1.5x volume ETOH
Apply to Omega Insect
Column
Extraction Method
Total yield (ng) ng into XT ng/ul out of XT
Mo Bio PowerSoil -B
Mo Bio PowerSoil -B
45.0
46.3
1.44
1.38
4.38
7.88
Prepman Phenol Mod
21.7
0.93
0.362
Prepman Phenol Mod
24.5
1.01
0.416
Omega Phenol Mod
8.8
~1
3.08
Omega Phenol Mod
Prepman-Qiagen
Qiagen Gentra Pure
Yeast and Bacteria
Qiagen Gentra Pure
Yeast and Bacteria
61.0
17.8
1.06
1.23
6.56
0.664
29.6
1.25
9.5
12.0
1.28
6.7
Epicenter Soil Master
7.8
0.82
1.83
Epicenter Soil Master
CTAB
CTAB
Mo Bio PowerSoil -A
Mo Bio PowerSoil -A
Sigma Extract-N-Amp
Tissue
Sigma Extract-N-Amp
Tissue
39.4
195.0
327.0
184.0
151.0
0.79
1.12
1.25
1.15
1.01
5.44
2.76
4.7
5.58
5.42
NA
1
2.88
NA
0.96
2.96
Library Preparation
• Nextera Library Construction mini experiment
– Vary numbers of PCR cycles (6, 12) needed for
adapter ligation of barcodes
– Pooled libraries were run on as a single sample and
deconvoluted during analysis (MiSeq)
– Determine the least number of cycles necessary for
reliable output - 12
• Illumina Nextera XT (standard protocol)
• 0.79 to 1.44ng of extracted DNA input
• Manually pooled barcoded samples based on
Qubit and Bioanalyzer measurements
Cluster generation and sequencing on
the HiSeq 2500
• Normalized pool to 27 nmoles, denatured with 2N
NaOH, diluted and loaded a final concentration of 3.0pM
• Cluster generation was performed on-board the HiSeq
• Catalog numbers:
- TruSeq Rapid SBS kit - HS (200 cycle) - Cat# FC-402-4001
- TruSeq Rapid PE Cluster Kit - Cat# PE-402-4001
• Sequencing was performed as a 100bp paired-end “Rapid”
run
• Run time on the HiSeq 2500: 27hrs!!!!!
Analysis
• Bowtie v 3-best-M1
– 11 genomes or close relatives totaling 53 Mbases
– Counted crude fractions that match
• >97% identical to template
– Divide number of match reads by genome size
– Duplicates averaged
• The genome sequence for Klebsiella
terrigena and Sporosarcinia ureae are not
available to do the comparable analysis.
Prepman Qiagen
Bacillus cereus
MB Power-A
Bacillus
megaterium
Prepman phenol
Epicenter Soil
Stapylococcus
epidermidis
Omega Phenol
Streptomyces
griseus
CTAB modified
Sigma Red Extract
Enterococcus
faecalis
MB Power-B
Micrococcus luteus
Qiagen Y&B
0
0.2
0.4
0.6
0.8
1
Prepman Qiagen
MB Power-A
Prepman phenol
Pseudomonas
aeruginosa
Epicenter Soil
Omega Phenol
Rhodospirilium
rubrum
CTAB modified
Enterobacter
aerogenes
Sigma Red Extract
MB Power-B
Qiagen Y&B
0
0.2
0.4
0.6
0.8
1
Reads per Organism for Extraction
Procedures
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
Stapylococcus epidermidis
(GC 32%, Gram +)
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
Micrococcus luteus
(GC 72%, Gram +)
1000000
800000
600000
400000
200000
0
600000
1200000
1000000
800000
600000
400000
200000
0
Enterococcus faecalis
(GC 38%, Gram +)
Streptomyces griseus
(GC 72%, Gram +)
2500000
2000000
1500000
1000000
500000
0
400000
Prepman…
Enterobacter aerogenes
(GC 53%, Gram -)
MB Power-A
Prepman…
Epicenter…
Omega…
Modified…
6000000
5000000
4000000
3000000
2000000
1000000
0
Sigma Red…
Rhodospirilium rubrum
(GC 64%, Gram -)
Qiagen Y&B
Prepman…
0
MB Power-B
MB Power-A
Prepman…
Epicenter…
Omega Phenol
Prepman…
MB Power-A
Prepman…
Epicenter…
Modified…
Modified…
Omega Phenol
Sigma Red…
Qiagen Y&B
Qiagen Y&B
Sigma Red…
MB Power-B
MB Power-B
200000
Pseudomonas aeruginosa
(GC 67%, Gram -)
2000000
1500000
1000000
500000
0
Bacillus cereus
(GC 35%, Gram +)
10000000
7500000
5000000
2500000
0
MB Power-B
Qiagen Y&B
Sigma Red…
Modified…
Omega…
Epicenter…
Prepman…
MB Power-A
Prepman…
Bacillus megaterium
(GC 38%, Gram +)
Conclusions
• Not all extraction techniques are created equal
for bacteria
• Column-based extraction may contribute to
reduced recovery due to DNA fragment size and
column inconsistency
• The use of PEG 6000 in a precipitation step may
be advantageous to increased recovery
• Multi-enzyme digestion seem to facilitate a
“broader” range of bacteria that gets extracted
but does not help total recovery in this study
• GC content, library prep and sequencing platform
must also be considered
Acknowledgments
Rachel Yoho (Ohio University Genomics Facility),
Marcy Kuentzel (UAlbany Center for Functional Genomics)
Lydia Zeglin (Oregon State University)
Mehmet Balkan (Portland State University)
Amy Janiak (Dana-Farber Cancer Institute)
Kendra Walton (Stowers institute)
Jim Vallandingham (Stowers institute)
Folker Meyer (Argonne National labs)
Will Trimble (Argonne National Labs)
Aimee Keithly (Illumina)
Vendor Acknowledgement
Integrated DNA Technologies
Illumina
Zymo
Omega Biotech
Qiagen
Epicenter Biotechnologies
LifeTechnologies
Mo Bio
Sigma
NARG Membership
Herb Auer
Nicholas Beckloff
Russ Carmical (Co-Chair)
Zach Herbert
Jennifer Holbrook (Co-Chair)
Vijay Nadella
Mark Robinson
Caprice Rosato
IRB Barcelona Spain
Case Western Reserve University
UTMB - Galveston
Dana-Farber Cancer Institute
Nemours Hospital for Children
Ohio University
University of Zurich
Oregon State Univ
Scott Tighe (Ad-Hoc-Outgoing)
Sridar V Chittur (Ad-Hoc-Outgoing)
Vermont Cancer Center
SUNY Albany
Anoja G Perera (EB liason)
Stowers Institute
New NARG Members
Drinks
(aka “Networking Events”)
are on Scott Tighe’s bar tab!