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 (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)(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

Microbe ATCC # Gram GC

Calculated as

Shipped

Bacillus megaterium 14581 + Rod Motile Spore forming 38 9.28E+06

Bacillus cereus 11778 + Rod Motile Spore forming 35 4.80E+06

Rhodospirillum rubra 9791 - Rod Purple nonsulfur phototrophic 64 9.28E+06

Sporosarcina ureae 13881 + Cocci Spore Forming 42 9.92E+06

Morphology

Sporosarcina ureae 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

Staphylococcus epidermidis 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

Streptomyces griseus 10137 + Filament Mycelia and terminal Spore forming 72 1.31E+06

1.08E+08Cultures were assembled to mathematically achieve 1.1 x108

Microbe Percent in Synthetic Cocktail

Bacillus

megaterium

9%

Bacillus cereus

4%

Rhodospirillum

rubra

9%Klebsiella

terrigena

9%

Micrococcus

luteus

9%

Streptomyces

griseus

1%

Sporosarcina

ureae

9%

Enterococcus

faecalis

9%

Pseudomonas

aeruginosa

7%

Enterobacter

aerogenes

11%

Staphylococcus

epidermidis

23%

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(duplicate samples), different procedures

modified

per manufactures recommendation

1 method (single sample)

TWEAK:

Sample added to Bead

Tube then homogenized

on bead beater 30

seconds at 4200RPM

REDExtract-N-Amp

Tissue PCR Kit

Transfer sample to 200ul PCR tubes

Add 20ul Extraction buffer (including

0.25 volumes of tissue prep solution)

Incubate RT 10 minutes

Mo Bio PowerSoil kit-B Mo Bio PowerSoil kit

adapted -A

w/Vortex Genie

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 CTABModified from K. DeAngelis et al.

Environmental Microbiology 12, 3137-

3149 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

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

Qiagen Gentra

PureYeast & Bacteria

Kit

Lyticase

Protease

Rnase A

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.

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.

NOTE: This reagent is not designed

for metagenomics. It is intended for

simple “lyse-n-amp” of a single

bacterial culture-

• Add 200ul of PrepMan

Ultra

• Add 350ul of PCI

• 5 sec COVARIS (20%-

PrepMan® Ultra Kit

Modified Modified Omega Insect

Kit

• Add 350 PCI and Vortex

1min

• Spin and Xfer supernatant

to new tube

• Add equal amount of

Omega CBL and follow SOP

Modified PrepMan®

Ultra with Phenol and

SDS

Sample Prep1. Multi-enzyme digestion 37oC

5 hrs +O/N RT

ReadyLyse – 2400U

Mutanolysin - 7U

Achromopeptidase- 1200U

Lysostaphin - 8U

Lysozyme - 100ug

Lyticase - 30U

Chitinase -100U

2. Boil 5 min

3. Proteinase K digestion • 5 sec COVARIS (20%-

10Int-1000b)

• Spin and Xfer

supernatant

• Add 250ul Chloro:IAA-

Spin- Xfer Supernatant

• Add 1.5x volume ETOH

• Apply to Omega Insect

Column

SDS• Add 200ul of PrepMan

Ultra

• Add 5% SDS

• 5 sec COVARIS (20%-

10Int-1000b)

• Add 250ul Chloro:IAA-

Spin- Xfer Supernatant

• Add 2x volume ETOH

• Apply to Qiagen Gel

band extraction column

3. Proteinase K digestion

(20mg/ml) 37oC 5hrs +RT O/N)

4. Add Ceramic Ball (MP bio) and

100mg of 1mm Diamond:ALO3

abrasive (200um)

5. FastPrep 4K rpm 30 sec

Extraction Method Total yield (ng) ng into XT ng/ul out of XTMo Bio PowerSoil -B 45.0 1.44 4.38

Mo Bio PowerSoil -B 46.3 1.38 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 61.0 1.06 6.56

Prepman-Qiagen 17.8 1.23 0.664

Qiagen Gentra PureYeast and Bacteria 29.6 1.25 9.5Yeast and Bacteria 29.6 1.25 9.5

Qiagen Gentra Pure Yeast and Bacteria 12.0 1.28 6.7

Epicenter Soil Master 7.8 0.82 1.83

Epicenter Soil Master 39.4 0.79 5.44

CTAB 195.0 1.12 2.76

CTAB 327.0 1.25 4.7

Mo Bio PowerSoil -A 184.0 1.15 5.58

Mo Bio PowerSoil -A 151.0 1.01 5.42

Sigma Extract-N-Amp Tissue NA 1 2.88

Sigma Extract-N-Amp Tissue 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 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.

0 0.2 0.4 0.6 0.8 1

Qiagen Y&B

MB Power-B

Sigma Red Extract

CTAB modified

Omega Phenol

Epicenter Soil

Prepman phenol

MB Power-A

Prepman QiagenBacillus cereus

Bacillus

megaterium

Stapylococcus

epidermidis

Streptomyces

griseus

Enterococcus

faecalis

Micrococcus luteus

0 0.2 0.4 0.6 0.8 1

Qiagen Y&B

MB Power-B

Sigma Red Extract

CTAB modified

Omega Phenol

Epicenter Soil

Prepman phenol

MB Power-A

Prepman Qiagen

Pseudomonas

aeruginosa

Rhodospirilium

rubrum

Enterobacter

aerogenes

0200000400000600000800000

1000000120000014000001600000

Bacillus megaterium

(GC 38%, Gram +)

0

200000

400000

600000

800000

1000000

Bacillus cereus

(GC 35%, Gram +)

0200000400000600000800000

10000001200000

Micrococcus luteus

(GC 72%, Gram +)

Reads per Organism for Extraction

Procedures

Streptomyces griseus

(GC 72%, Gram +)3500000

Stapylococcus epidermidis

(GC 32%, Gram +)600000

Enterococcus faecalis

(GC 38%, Gram +)

0500000

1000000150000020000002500000

0500000

100000015000002000000250000030000003500000

MB

Pow

er-B

Qia

gen

Y&

B

Sig

ma R

ed …

Mod

ifie

d …

Om

ega P

hen

ol

Ep

icen

ter …

Pre

pm

an

…

MB

Pow

er-A

Pre

pm

an

…

0

200000

400000

600000

0250000050000007500000

10000000

MB

Po

wer

-B

Qia

gen

Y&

B

Sig

ma R

ed …

Mo

dif

ied

…

Om

ega …

Ep

icen

ter …

Pre

pm

an

…

MB

Po

wer

-A

Pre

pm

an

…

Enterobacter aerogenes

(GC 53%, Gram -)

0100000020000003000000400000050000006000000

MB

Pow

er-B

Qia

gen

Y&

B

Sig

ma R

ed …

Mod

ifie

d …

Om

ega …

Ep

icen

ter …

Pre

pm

an

…

MB

Pow

er-A

Pre

pm

an

…

Rhodospirilium rubrum

(GC 64%, Gram -)

0500000

100000015000002000000

MB

Pow

er-B

Qia

gen

Y&

B

Sig

ma R

ed …

Mod

ifie

d …

Om

ega P

hen

ol

Ep

icen

ter …

Pre

pm

an

…

MB

Pow

er-A

Pre

pm

an

…

Pseudomonas aeruginosa

(GC 67%, 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 • 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) 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

QiagenQiagen

Epicenter Biotechnologies

LifeTechnologies

Mo Bio

Sigma

NARG Membership

Herb Auer IRB Barcelona Spain

Nicholas Beckloff Case Western Reserve University

Russ Carmical (Co-Chair) UTMB - Galveston

Zach Herbert Dana-Farber Cancer Institute

Jennifer Holbrook (Co-Chair) Nemours Hospital for Children

Vijay Nadella Ohio University

Mark Robinson University of ZurichMark Robinson University of Zurich

Caprice Rosato Oregon State Univ

Scott Tighe (Ad-Hoc-Outgoing) Vermont Cancer Center

Sridar V Chittur (Ad-Hoc-Outgoing) SUNY Albany

Anoja G Perera (EB liason) Stowers Institute

New NARG Members

DrinksDrinks

(aka “Networking Events”)

are on Scott Tighe’s bar tab!