Technical Strategies for Oxford Nanopore Sequencing and ...
Transcript of Technical Strategies for Oxford Nanopore Sequencing and ...
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Scott Tighe
University of Vermont Cancer CenterAdvanced Genomics Core
ABRF Metagenomics Research Group
Technical Strategies for Oxford Nanopore Sequencing and Rapid Pathogen Detection and Biosurveillance
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• Steering Committee of IMMSA• Chair ABRF Metagenomics Research Group • Leader Extreme Microbiome Project (50 members)• Member MetaSub International Consortium• Member Genomics Standards Consortium
Background:Microbiologist, Mycologist, Phycologist, Molecular BiologistNext generation sequencing facility manager
Oxford Nanopore, Illumina HiSeq
Cultured and identified tens of thousands of bacteria, fungi, parasitesusing growth, CFA, Biolog, MIS-MIDI, microscopy, ect
Started working in Microbiome analysis in 1984 at Northern Arizona Univ
Contact Info [email protected]
Speaker Info
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The ABRF Metagenomics Research is a team devoted to study and improve methods and consumables used in metagenomics research
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The XMP is a proving ground for studying consumables on the most challenging of sample types
As well as collect shotgun data on novel sample sites
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• Rapid Pathogen Screening has its Challenges
• Many sample types have limited DNA
• Require some amplification Swabs, drinking water, food wash, ect
• Bacteria are difficult to lyse uniformly
• Lysis slurries often inhibit PCR reactions and other synthesis reactions
Oxford Nanopore Sequencing and Rapid Pathogen Detection and Biosurveillance
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Recent Innovations Enable Rapid Lysis and Amplification
• Metapolyzyme• PrepMan Ultra™• Unreactive beater beads-Diamond• High performance Titanium Taq Polymerase• Modified SKQ-RAB201 Oxford Nanopore
Protocol
Swab Technique modified from Mason et al MetaSub International Metagenomics Consortium
Originally designed for rapid screening of Legionella pneumophilia
The Goal: Swab to Sequence in 60 minutes
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Workflow for Rapid Bacterial Screening
150 ng input to SQK-RAB201
25 cycles
Use Clontech/Takara Titanium Taq
1-4ul input 16s PCR
100C 10 min -Vortex
Add 10-100ul of Prepman Ultra™
Add 4ul MetaPolyzyme 35C 20 min
Swab surface
100ul PBS pH7.5 with diamond Beads
50ul Titanium Taq PCR95/1:0095/3054/30 25cyc68/1:3068/3:00
35C 20m 100C 10m
Ampure Beads
4ul
ONT
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Bacteria and Fungi have a Cell Wall and are Difficult to Lyse
DNA extraction efficiency studies DNA extraction Study with 12 bacteria at known concentration by the ABRF Nucleic Acids Research
group in 2011. Whole cell microbial standard -ETOH fixed Microscopically counted
ABRF Nucleic Acids Research Group 2012-2013 StudyEvaluating DNA Extraction Methods for Metagenomic Analysis
V. Nadella1, J. Holbrook2, R. Carmical3, M. Robinson4, C. Rosato5, H. Auer6, N. Beckloff7, Z. Herbert8, S. Chittur9, A. Perera10 , W. Trimble11, S. Tighe121Ohio University, 2Nemours/A.I. DuPont Hospital for Children, 3University of Texas Medical Branch, 4 University of Zurich, Switzerland, 5Oregon State University, 6Institute for Research in Biomedicine, Barcellona, Spain, 7Case Western Reserve University, 8Dana Farber Cancer Institute, 9University at Albany-SUNY, 10 Stowers Institute for
Medical Research, 11Argonne National Laboratory, 12University of Vermont.
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Chemical and beads alone don’t lyse all cells
Control CTAB Phenol
SDS CTAB+Beater Beads CTAB+Phenol+Beader
Micrococcus luteus lysis vs chemical and Beater Beads
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New Innovations=New Approaches
MetapolyzymePrepMan Ultra Titanium TaqOxford Nanopore sequencerRapid Analysis Software
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35C pH 7.5
Use alone or combined with Lysozyme
REQUIRES PBS pH 7.5 and NO EDTA
Designed specifically for Bacterial, Fungi, and Yeast
Originally formulated in 2004
Tested in 2016 by ABRF MGRG
Commercialized in 2017 by Millipore Sigma
Why Develop Metapolyzyme?Lysis without beater beads or aggressive vortexing. If you can sphearoplast all the cells,
Proteinase K and Detergent can do the rest.
Metapolyzyme
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Sphearoplasting is Key
• Multi-lytic Enzyme Mix for Digestion of Cell Walls
MetaPolyzyme
Bacteria, yeast, fungi cell wallsPhylozyme (3 additional
enzymes for Plant and Algae)Testing Started 11/2016
Micrococcus luteus
No Enzyme Enzyme
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020406080
100120140160
ng/u
l(no
rmal
ized)
Polyzyme No Enzyme
Beta Test Results
• Over 150 sample trials (Polyzyme, PBS only, Polyzyme only)
• 3 trials with Lysozyme alone-Need more data
• 6 labs , 17 matrices• Any kits
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How Low and Fast can Sphearoplasting Occur?
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
No Poly 0.6uL 1.3ul 2.5ul 5ul 10ul 20ul PBS
Metapolyzyme Titration1hr 3hr 5hr Nano Qubit
20 0.6, C
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Rapid cell lysis reagent100c 10minUsed for ABI’s MicroSeq ID systemBacteria and fungiVery little inhibition on downstream reactions
2-ButoxyethanolSodium metasilicateCitric AcidBuffers
PrepMan Ultra™
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Diamond Beater Beads
• Beater Beads Fragment DNA• Not ideal for Oxford Nanopore but ok for full length 16s• Not desirable when using long read sequencers• Mild use for 16s full amplicon-1492bp • Synthetic Diamond is unreactive• Limited vortexing
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Workflow for Rapid Bacterial Screening
150 ng input to SQK-RAB201
25 cycles
Use Clontech/Takara Titanium Taq
1-4ul input 16s PCR
100C 10 min -Vortex
Add 10-100ul of Prepman Ultra™
Add 4ul MetaPolyzyme 35C 20 min
Swab surface
100ul PBS pH7.5 with diamond Beads
50ul Titanium Taq PCR95/1:0095/3054/30 25cyc68/1:3068/3:00
35C 20m 100C 10m
Ampure Beads
4ul
ONT
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Successful PCR is the Key
• Tested 6 high performance Taq polymerases• Life Phusion• NEB Q5• KAPA HIFI• Takara Ex-Taq• ABI Gold MM• Takara Titanium
• Mixed with different amounts of lysis material
• Different input of samples• Used Oxford Nanopore 16s Barcode primers.
• 1492 rev• 27 Fwd -Single degenerate-Not optimal
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Comparison of Taq Polymerase in the Presence of MetaPolyzyme and Prepman Ultra
16s Full Length PCR on Rapid Sample Oxford Nanopore Primers –SQK-RAB201
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Oxford Nanopore Sequencing
Use of the 16s Rapid Barcoding Kit (SQK RAB201)
10 ng input gDNA (normally) 27f (1d) and 1492r PCR step Barcoding of 12 samples Modified PCR step using Clontech Titatium Taq (639209) 25 cycles Requires 100 ng input of full length 16s Amplicon library into the
sequencer
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ONT Sequencer Stats
• R9.4 (893 pores)**• 35,000 reads in 50 minutes• 150 ng input library • 500,000 reads in 14 hrs
** Flow cell traveled to Antarctica and back and was 6 months old
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QC Output for Oxford RAB201-TiTaq
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Results from Rapid Analysis Software
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What’s Next
• Live vs Dead• Modified Nextera/Transposase WGS • Unk-ome or dark matter
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Acknowledgements
The ABRF MGRG Team
The Extreme Microbiome Team
Michael Micorescu Oxford Nanopore
Sarah Johnson Georgetown
Sam Greenfield-UVM
Applied Biosystems
Chris Mason and Noah Alexander-Weill Cornell
Scott Jackson and Jason Kralj -NIST
Cosmos ID- Rita, Manoj, Nur, Huai
One Codex-Nick Greenfield
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Rapid Field Sequencing in Antarctica using the Oxford Nanopore Sequencer
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The Sarah Johnson team (AKA G062M)Sarah Johnson (PI), Angela Bai, David Goerlitz, Scott Tighe, Elena
Zaikova
NSF EAGER grant: Single-Molecule Sequencing of Antarctic Paleolakes
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Antarctica Dry Valleys
Magnetic South Pole
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Glacial Lake VictoriaLong gone but microbes remain that we can sequence
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Collecting The Ancient Microbial Biofilms using Nucleic acid free sampling
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DNA Extraction Results
Agilent 2200 genomic tape
Agilent BA2100 HS Chip
Comparison of Column vs Bead capture. Column reduces DNA size
Average Yield from Victoria land samples
15-60kb
>10kb
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Field Deployable Oxford Nanopore Minion Sequencers
66 70 79 84 90
507090
Sprin
g
2015
Sum
me
r 201
5Fa
ll 20
15W
inte
r 20
16Sp
ring
2016
Oxford Nanopore Sequencing Accuracy
(%)
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DNA Sequencing in the Field• Library prep in the field with hot water incubator
• Sequencer kept warm with hand warmers
• Proof of principle for Exoplanet grant
• The dry valleys are the perfect Mars analog site
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37
Field Sequencing
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First Field : Taylor Valley Antarctica
Read length and nucleotide distribution for the MK1B MinION during field tests in the Taylor Valley, Antarctica.
Partial classification of reads collected in the field analyzed using One Codex Software. Interesting but questionable
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Compiled Runs Results
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Acknowledgements
The ABRF MGRG Team
The Extreme Microbiome Team
Michael Micorescu Oxford Nanopore
Sarah Johnson Georgetown
Sam Greenfield-UVM
Applied Biosystems
Chris Mason and Noah Alexander-Weill Cornell
Scott Jackson and Jason Kralj -NIST
Cosmos ID- Rita, Manoj, Nur, Huai
One Codex-Nick Greenfield