NIGMS funded Center

Experimental Methods
in Systems Biology
Part of the Coursera Certificate in Systems Biology
Marc Birtwistle, PhD
Department of Pharmacology & Systems Therapeutics
Fall 2014, Week 2, Deep mRNA Sequencing

Outline

PurposeofmRNAsequencing
LimitationsofTheFirstOmicsTechnologyMicroarrays
First,Second,andThirdGenerationSequencingTechnologies
FocusonIllumina (Secondgeneration)
ObtainingmRNA
Creatingalibrary
Pairedendvs.singleend
Ligate adaptors
AmplifybyPCR

Sequencing
Examplesofresults

Quantification
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WhatisthePurposeofmRNAseq?
mRNAsequencingallows:
Quantificationofallexpressedtranscriptlevels
Therecanbeissueswithhowquantitativeitisbecause
ofPCRstepsinsometechnologies

Quantificationofratiosofsplicevariantlevels
Identificationofnovelsplicevariants
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TheBigPicture:Applications of Next
GenerationSeq technologies

ImpactonResearch:
Redefinesquestionsresearcherscanask

From'DoesthisproteinregulategeneX?'to'Whatgenes
doesproteinXregulate?'
From'DoesthisgenecausediseaseX?'to'What
differencesinthegenomecauseddiseaseX?'

Newapplicationsbeingdevelopedrapidly Plentyof
opportunitytobecreative!

TheFirstTranscriptomics TechnologyMicroarrays

Microarraybasedtechnologiesare
currentlycheaper(butprobablynotfor
long)
Disadvantages
Resultsaredifficulttocompareacross
platforms(i.e.differentmicroarray
companies)
Youhavetoknowwhatyouarelooking
for

Hardtoidentifynoveltranscriptsorsplice
variants

Quantifyingalternativesplicingisdifficult
(butnotimpossible)

http://www.genome.gov/10000533

Inthelongrun,thepuresequence
dataprovidedbymRNAsequencing
willlikelyprovemorereliableandeasy
toshare
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EvolutionofSequencingTechnologies

http://www.genome.gov/images/content/cost_per_genome2.jpg
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FirstgenerationSangersequencing
Reaction

Size

Readout

http://en.wikipedia.org/wiki/Sanger_sequencing
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BenefitsanddrawbacksofSanger
sequencing
Throughput:
Read length:
Chemistry:
Advantage:
Drawbacks:

One strand at a time

~700 bp
Chain termination
Highly accurate
Very slow and expensive (on a genome scale)

IncreasingDNASequencing
throughput

Sequencing many many DNA strands at a time to increase throughput

Automation of Sequencing process

96 and 384 well format for Sanger sequencing was developed.

Fluorescent labeling of ddNTPs and capillary electrophoresis developed
that enabled automation of Sanger sequencing.
Isthisincreasesufficient?
Even with increase in throughput and automation, it took
13 years to complete 1 human genome sequencing at the cost of ~$2.7
billion!
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NeedtoincreaseDNAsequencingby
severalorderofmagnitude
Increasenumberofwellsperlocationfromexistingmaximumof384wellplate
Literallygodowntomicro/nano wellplateformat.
Increasetoaccommodateasmanyasmillionandmorewellsperplate!!

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Canweusedd Chainterminationchemistry
tosequenceDNAin>millionmicrowell format?
Sangersdd chainterminationchemistrygeneratesseverallabeled
DNAstrandsthatnecessitatesseparationofDNAstrandsgeneratedin
incrementofonebaselengthperBaseofsequence.

So,weneedtothinkofdifferentsequencingchemistrythatdoesnot
requireDNAseparationstep.

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