the importance of dna extraction in …...the importance of dna extraction in metagenomics: the...
TRANSCRIPT
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
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0250000050000007500000
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Enterobacter aerogenes
(GC 53%, Gram -)
0100000020000003000000400000050000006000000
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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!