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

1.250l of Soil DNA Extraction buffer + 2l of

Proteinase K; vortex briefly.
2. Shake the tube at 37C for 10 minutes.
3. Add 50l of Soil Lysis Buffer and vortex
briefly.
4. Incubate at 65C for 10 minutes.
5. Add 60l 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 6l 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 500l 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 12l 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

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

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

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

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 Tighes bar tab!