CS82008 AugusL 2008 uCSC Sequenclng CenLer

1) repare AdapLer LlgaLed ssunA Llbrary (A-[lnserL]-8) 2) LmC8: Clonal Ampllcauon on 28

beads followed by enrlchmenL
4) erform sequenclng-by-synLhesls
on Lhe 434 Sequencer
3) Load beads and enzymes
ln lco1lLer laLe`
Cvervlew of 1he 434 Sequenclng SysLem
CS82008 AugusL 2008 uCSC Sequenclng CenLer
M|x DNA L|brary
& capture beads
(||m|ted d||unon)
Lmulslon 8ased Clonal Ampllcauon
"8reak m|cro-reactors"
Iso|ate DNA conta|n|ng beads
- Cenerauon of mllllons of clonally amplled sequenclng LemplaLes on each bead
- no clonlng and colony plcklng
Create
"Water-|n-o||"
emu|s|on
+ Ck keagents
+ Lmu|s|on C||
erform emu|s|on Ck
Adapter carry|ng
||brary DNA
A
8
M|cro-reactors
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Centrifuge Step
Load Lnzyme
8eads
44 m
Load beads |nto
|co1|ter|ate
ueposlung unA 8eads lnLo Lhe lco1lLer`laLe
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng 8y SynLhesls
unA CapLure 8ead
ConLalnlng Mllllons of
Coples of a Slngle
Clonal lragmenL
A A 1 C G G C A 1 G C 1 A A A A G 1 C A
Annea| r|mer
! SlmulLaneous sequenclng of Lhe enure genome ln hundreds of
Lhousands of plcollLer-slze wells
! yrophosphaLe slgnal generauon
1

|
A1
L|ght + oxy |uc|fer|n
Su|fury|ase
Luc|ferase
AS
|uc|fer|n
Sequenc|ng-8y-Synthes|s
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng Workow Overview
Clonal amplification of fragments bound to
beads in microreactors
One Bead
One Read
400,000 reads
per run
One Fragment
Generation of small DNA fragments via
nebulization
Ligation of A/B-Adaptors flanking single-
stranded DNA fragments
Emulsification of beads and fragments in
water-in-oil microreactors
Sequencing and base calling
Sample input: Genomic DNA, BACs,
amplicons, cDNA
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng Workow
Library Preparation
8.0 h 7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration emPCR Sequencing
sstDNA library created with adaptors
A/B fragments selected using streptavidin-biotin
purification
Cenome fragmenLed by
nebullzauon
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng Workow
Emulsion PCR
8.0 h 7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration
emPCR Sequencing
Emulsion-based clonal amplification
Anneal sstDNA
to an excess of
DNA Capture
Beads
Emulsify beads
and PCR
reagents in
water-in-oil
microreactors
Break
microreactors,
enrich for DNA-
positive beads
Clonal amplification
occurs inside
microreactors
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng Workow
Loading of PicoTiterPlate Device
Well dlameLer: average of 44 m
> 400,000 reads obLalned ln parallel
A slngle clonally amplled ssLunA
bead ls deposlLed per well
Depositing DNA beads into the PicoTiterPlate device

Quality filtered bases Amplified sstDNA library beads
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng Workow
Sequencing by Synthesis
8ases (1ACC) are owed sequenually and
always ln Lhe same order (100 umes for a
large CS lLx run) across Lhe
lco1lLerlaLe devlce durlng a
sequenclng run.
A nucleoude complemenLary Lo Lhe
LemplaLe sLrand generaLes a llghL slgnal.
1he llghL slgnal ls recorded by Lhe CCu
camera.
1he slgnal sLrengLh ls proporuonal Lo Lhe
number of nucleoudes lncorporaLed.
8.0 h 7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration
emPCR Sequencing
Flowgram
Key sequence
CS82008 AugusL 2008 uCSC Sequenclng CenLer
CS lLx uaLa Analysls
Flowgram Generation
!"#$%&'(
Key sequence = TCAG for signal calibration
llow Crder
1-mer
2-mer
3-mer
4-
mer
1
A
C
G
11C1GCGAA
CS82008 AugusL 2008 uCSC Sequenclng CenLer
CS lLx uaLa Analysls
Overview
GS De Novo Assembler
GS Reference Mapper
GS Amplicon Variant Analyzer
Image capture
Image processing
Signal processing GS Run Browser
CS82008 AugusL 2008 uCSC Sequenclng CenLer
CS lLx SysLem erformance
Read Length
CS82008 AugusL 2008 uCSC Sequenclng CenLer
1he Genome |s compr|sed of repeat reg|ons
uependlng upon Lhe speclc genome characLerlsucs,
mlcroreads (~23 bp's) cover only a poruon of Lhe genome
ln human - 23 base palr reads can only be mapped
unlquely Lo 80 of Lhe genome
ShorL reads are llmlung ln known genomes - WhaL
abouL unknown genomes?
Mapplng versus de novo assemblles
Mapplng wlll mlss genome rearrangemenLs
Mapplng ls only as good as Lhe reference
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Why Does Length Mauer?
Longer sequenclng reads mean more appllcauons
ldenufy and characLerlze small and shorL 8nA's
lull lengLh cunA sequenclng for expresslon levels and varlauons
Ampllcon resequenclng for geneuc varlauon lncludlng somauc muLauons
Sequenclng of mlcro-organlsms ln a slngle lnsLrumenL run
Sequenclng of complex genomes - mammallan & planL
Sequenclng of complex samples - MeLagenomlcs, AnclenL unA
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Longer sequenc|ng reads mean more app||canons
Plv SLudles (3)
Chl-Sequenclng (8)
8oyle eL al, Cell: Mlxed Lechnologles for mapplng open chromaun
MeLagenomlcs (12)
alaclos eL al, new Lngland !ournal of Medlclne, aLhogenlc vlrus ueLecuon
Whole Cenome Sequenclng (30)
velasco eL al, LoS: lnoL nolr Cenome
alred-Lnd sequenclng
ueLecung SLrucLural varlauons across Lwo human genomes
1echnology and 8lolnformaucs (11)
Meyer eL al, nA8,: uslng lcogram quanuues of sample
1ranscrlpLome sLudles - cunA (17)
Small 8nA (32)
Ampllcon and MeLhylauon SLudles (9)
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Appllcauons of Whole Cenome, ulLra 8road
and ulLra ueep P1- Sequenclng
n1- Sequenc|ng
1echno|ogy
App||canons
Who|e Genome
Sequenc|ng
V|rus
8acter|a
Iungus
n|gher Lukaryotes
numan
U|tra Deep
Sequenc|ng
opu|anon 8|o|ogy
nIV
8acter|a| 16S
kes|stance
1rop|sm
Amp||cons
U|tra 8road
Sequenc|ng
Sma|| kNA
Metagenom|cs
Lxpress|on
Nove| stra|n ID
1ranscr|ptome
nLA 1yp|ng
CS82008 AugusL 2008 uCSC Sequenclng CenLer
1he power of Metagenom|cs
Pow Lo ldenufy an envlronmenL based upon Lhe mlcroblal organlsms LhaL are presenL
Mlcroblal opulauon SLrucLures ln Lhe ueep Marlne 8losphere
!"#$% $' ()*+ ,-.$/-$+ 012+ 345+ 6775
ueLermlnlng Lhe sLaLe of an envlronmenL based upon Lhe presence and mlxLure of mlcroblal
organlsms
1he lnLerdependence of Coral and lL's mlcroblal envlronmenL
8$9)$: $' ()*+ ;/<.%=/>$/'() ?.-%=#.=)=9:+ 4+ 36575+ 6775
ueLecung vlral paLhogens - qulckly and accuraLely
Less Lhan 12 monLhs from rsL ldenucauon of aecLed hlves Lo posslble paLhogen
@=ABC=D'$% $' ()*+ ,-.$/-$+ 6775
1ransplanL vlcums from AusLralla
E()(-.=D $' ()+ F$G ;/9)(/H I="%/() =J ?$H.-./$+ 6772
CS82008 AugusL 2008 uCSC Sequenclng CenLer
1ranscrlpLome Analysls
Workflow Comparison
GS FLX (clonal sequencing ensured through emPCR)
Sanger (E. coli cloning, often concatemerization)
cDNA libraries
(short tag library,
EST library)
Concatemerization,
insert fragments into vectors
and clone into bacteria
Grow, pick colonies
Template Generation
Sequencing
Time: Weeks
emPCR
Sequencing
Time: Days
cDNA libraries
(short tag library,
EST library)
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenclng of approxlmaLely 400,000 small
8nAs from @* $)$9(/D
AnoLher 18 unknown ml8nA genes were
deLecLed
1housands of endogenous sl8nAs acung
preferenually on LranscrlpLs assoclaLed wlLh
spermaLogenesls and Lransposons were
ldenued
A new class of small 8nAs was ldenued: 21U-
kNAs. 1hey all begln wlLh an u and are
preclsely 21 nL long.
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Muluplex ldenuer Basics
WhaL ls lL?
1wo new k|ts, each wlLh 6 dlerenL llbrary adapLers (LoLal of 12 adapLers)
Lach Mlu llbrary adapLer has an added, speclally encoded 10-base reglon
used Lo bar-code" up Lo 12 dlerenL genomlc llbrary samples Lo be run ln Lhe
same reglon of a slngle sequenclng run
r|mer A MID 1 key L|brary fragment r|mer 8
Seq. pr|mer kead
#bases: 1S 4 10
r|mer A key L|brary fragment r|mer 8
#bases: 40 4
MID
L|brary
Standard
L|brary
Seq. pr|mer kead
r|mer A MID 2 key L|brary fragment r|mer 8
r|mer A MID n key L|brary fragment r|mer 8
CS82008 AugusL 2008 uCSC Sequenclng CenLer
alred-Lnd Appllcauons
~100 bp sequenclng Lags separaLed by 3 kb spaclng
use for de novo assembly
Crder conugs
use for SLrucLural varlauon sLudles
lnverslons, ueleuons, lnseruons.
Plgh resoluuon deLecuon - 3kb spaclng vs 10 Lo 40 kb
CS82008 AugusL 2008 uCSC Sequenclng CenLer
a|red-Lnds workow
CS82008 AugusL 2008 uCSC Sequenclng CenLer
1argeted Lnr|chment of numan gDNA
gunA
Lxon 1 Lxon 2 Lxon 3 Lxon 4 Lxon S
lragmenL and hybrldlze Lo
nlmbleCen capLure array
P1-Sequenclng
Analyze
Lxon
Sequences
LluLe
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Sequenc|ng a|| the known exons from the human
genome
ulrecL selecuon of human genomlc locl by mlcroarray
hybrldlzauon,"
AlberL eL al., naLure MeLhods, (4) 11, 903 -903, 2007
~6,700 gunA locl selecLed
88CA1 reglon
2 MB Region
CS82008 AugusL 2008 uCSC Sequenclng CenLer
Another Sequence-Capture Lxamp|e
19 kb reglon from Chromosome 4
GS ILk Seq keads
Sequenc|ng Coverage
Seq-Cap Array robes
1argeted Lxons