DNASequencing

GenomicScienceandLeadershipWorkshopMay25,2017

AgnesChan

(1)WhySequencingDNA?

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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

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WhystudyingDNAsequence?Studymicrobeslivinginnatureandourbodies(microbiome)

http://learn.genetics.utah.edu/content/gsl/diversity/ 4

Environments

Human skin microbiome

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

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WhystudyingDNAsequence?Decodethehumangenome:identifygenes,healthordisease

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SequenceandStructuralVariants

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WhystudyingDNAsequence?Decodethehumangenome:Geneticdiversitywithinandacrosspopulations

By Jthiele at English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=54137526

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(2)SequencingTechnology

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GrowthofDNASequencing

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.100219510

TheCostofSequencingperHumanGenome

https://www.genome.gov/sequencingcosts/11

SangerSequencing– ChainTermination

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http://www.cegat.de/web/wp-content/uploads/2015/10/sequencing-1.png 13

IlluminaSequencing– Sequencing-by-synthesis

IlluminaSequencing– BridgeAmplification

http://www.3402bioinformaticsgroup.com/wp-content/uploads/2016/07/NGS.png 14

IlluminaTechnologyVideo

• https://www.youtube.com/watch?v=HMyCqWhwB8E• ClusterGeneration• Sequencing-by-synthesis(0to3:09)

NanoporeSequencinghttps://nanoporetech.com/how-it-works

http://www.nature.com/nrg/journal/v17/n6/fig_tab/nrg.2016.49_F5.html16

• 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

(3)DNALabfromTuesday

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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

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Interpret DNA Sequence

DNA Sequencing (Illumina MiSeq)

Day 1

Day 3

Who’sthere?

http://learn.genetics.utah.edu/content/microbiome/study/

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Review16SPCRresultsfromDay1

Foreachgroup:1. DNAsizemarker(100bp ladder)2. Positivecontrol3. Negativecontrol4. Skateparkrunoff

100 bp

• WhatisthesizeofthePCRproduct?• Whatdeterminesitssize?

Computer Lab Day:Sample Analysis in QIIME

Joslynn Leejoslynnlee@gmail.com