dna sequencing - craig ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018....
TRANSCRIPT
![Page 1: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/1.jpg)
DNASequencing
GenomicScienceandLeadershipWorkshopMay25,2017
AgnesChan
![Page 2: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/2.jpg)
(1)WhySequencingDNA?
2
![Page 3: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/3.jpg)
What is DNA (Deoxyribonucleic acid)?A biomolecule containing genetic information.DNA sequencing: Determine the order of nucleotides of a DNA fragment
https://www.genome.gov/18016863/a-brief-guide-to-genomics/ 3https://www.genome.gov/images/content/dna_factsheet.j
pg
![Page 4: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/4.jpg)
WhystudyingDNAsequence?Studymicrobeslivinginnatureandourbodies(microbiome)
http://learn.genetics.utah.edu/content/gsl/diversity/ 4
Environments
Human skin microbiome
![Page 5: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/5.jpg)
WhystudyingDNAsequence?Decodethehumangenome:Firsthumangenomesequenced(1990-2003),1000humangenomes
By Jthiele at English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=5413
75265
![Page 6: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/6.jpg)
WhystudyingDNAsequence?Decodethehumangenome:identifygenes,healthordisease
6
![Page 7: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/7.jpg)
SequenceandStructuralVariants
7
![Page 8: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/8.jpg)
WhystudyingDNAsequence?Decodethehumangenome:Geneticdiversitywithinandacrosspopulations
By Jthiele at English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=54137526
8
![Page 9: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/9.jpg)
(2)SequencingTechnology
9
![Page 10: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/10.jpg)
GrowthofDNASequencing
http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.100219510
![Page 11: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/11.jpg)
TheCostofSequencingperHumanGenome
https://www.genome.gov/sequencingcosts/11
![Page 12: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/12.jpg)
SangerSequencing– ChainTermination
12
![Page 13: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/13.jpg)
http://www.cegat.de/web/wp-content/uploads/2015/10/sequencing-1.png 13
IlluminaSequencing– Sequencing-by-synthesis
![Page 14: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/14.jpg)
IlluminaSequencing– BridgeAmplification
http://www.3402bioinformaticsgroup.com/wp-content/uploads/2016/07/NGS.png 14
![Page 15: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/15.jpg)
IlluminaTechnologyVideo
• https://www.youtube.com/watch?v=HMyCqWhwB8E• ClusterGeneration• Sequencing-by-synthesis(0to3:09)
![Page 16: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/16.jpg)
NanoporeSequencinghttps://nanoporetech.com/how-it-works
http://www.nature.com/nrg/journal/v17/n6/fig_tab/nrg.2016.49_F5.html16
![Page 17: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/17.jpg)
• 5-10Gb of DNA sequence data
• Ultra-long reads• Real time data• PC laptop
OxfordNanoporeTechnology
https://nanoporetech.com/products/smidgion 17
• Field-based sequencing not confined to labs
• Pathogen outbreaks• Environmental extremes
SmidgIONMinION
![Page 18: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/18.jpg)
(3)DNALabfromTuesday
18
![Page 19: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/19.jpg)
WaterMicrobiomeStudyWorkflow
Collect samples
Isolate DNA Collect microbes on filter membrane
PCR amplification of 16S rRNA gene
Agarose gel analysis of PCR products
Run 16S analysis
0.05
W15
W02
CS1
W16
W05
W14
W08
W09
W12
W11
W01
CS2
CE2
W10
W06
CE1
W07
W04
W13
W03
Interpret DNA Sequence
DNA Sequencing (Illumina MiSeq)
Day 1
Day 3
![Page 20: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/20.jpg)
Who’sthere?
http://learn.genetics.utah.edu/content/microbiome/study/
20
![Page 21: DNA Sequencing - Craig Ventermaize.jcvi.org/cellgenomics/outreach/2017/notes/lecture5... · 2018. 6. 21. · Water Microbiome Study Workflow Collect samples Collect microbes Isolate](https://reader035.vdocument.in/reader035/viewer/2022081604/604fbfb40412633d595152f1/html5/thumbnails/21.jpg)
Review16SPCRresultsfromDay1
Foreachgroup:1. DNAsizemarker(100bp ladder)2. Positivecontrol3. Negativecontrol4. Skateparkrunoff
100 bp
• WhatisthesizeofthePCRproduct?• Whatdeterminesitssize?