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Early experiences in amplicon sequencing using the Roche GS-FLX massively parallel DNA sequencer and its
application within a diagnostic laboratory
Louise Stanley
Northern Genetics Service
Newcastle Upon Tyne
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GS-FLX - Roche
Next Generation DNA Sequencer
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GS-FLX - Roche
Next Generation DNA Sequencer Based upon pyrosequencing
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GS-FLX - Roche
Next Generation DNA Sequencer Based upon pyrosequencing
Sequencing Reagents
Camera
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Work-flow
Library preparation Emulsion PCR – Clonal Amplification Sequencing Data analysis
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Library Preparation
PCR amplification Purification of products Quantification Dilution to 2 x 105 molecules/l
Alternative methods – e.g. array based pull down by NimbleGenTM
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Emulsion PCR
+
Binding
Emulsion Formation
PCR Amplification
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Emulsion PCR
+
Binding
Emulsion Formation
PCR Amplification
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Emulsion PCR
+
Binding
Emulsion Formation
PCR Amplification
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Emulsion PCR
+
Binding
Emulsion Formation
PCR Amplification
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Emulsion PCR
+
Binding
Emulsion Formation
Emulsion Breaking and Bead Enrichment
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Sequencing
Occurs on PicoTitre Plates Capacity of LR70 ~ 100 MB Split into 2, 4, 8 or 16 regions
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Sequencing
G
T
C
A
1
2
3
4
5
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Sequencing
G
T
C
A
G
1
2
3
4
5
G
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Sequencing
G
T
C
A
G T
1
2
3
4
5
G T
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Sequencing
G
T
C
A
G T C
1
2
3
4
5
G T
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Sequencing
G
T
C
A
G T C A
1
2
3
4
5
G T A A
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Sequencing
G
T
C
A
G T C A G
1
2
3
4
5
G T A A G
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Sequencing
G
T
C
A
G T C A G T
1
2
3
4
5
G T A A G T T T T
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Sequencing
G
T
C
A
G T C A G T C
1
2
3
4
5
G T A A G T T T T C C C
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Data Analysis
Sequencing reads aligned to reference sequence
Software searches for variants – presented in both tabular and graphical formats
Some manual inspection of sequence data recommended
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Data Interpretation
Length of amplicon sequence
Number of reads across amplicon
Variants
Variant Table
Consensus Global Alignment Plot
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Data InterpretationVariant Table
Consensus Global Alignment Plot
T>CC>T
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Mutation Detection
Reference Exon Mutation Male/Female
D85356 3 c.106_109delinsT M
D99519 9 c.833C>T F
D99520 31 c.4290_4291del M
D99521 36 c.5067_5068delCCinsG F
D99522 58 c.8668+1G>A M
D42687 66 c.9568C>T M
D99523 69 c.10086+1G>T F
7 patients with different mutations in dystrophin gene examined
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Mutation Detection
All mutations identified and called by software in variant tables
Investigated level of coverage required for mutation detection
Achieved by mixing patient libraries with mutations with WT library
[c.4290_4291del; male][c.833C>T; female]
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Mutation Detection
Number of Regions
Reads per Region
Mb per Region
Amplicons per Region
2 200 000 50 ~ 3300
4 70 000 17.5 ~ 1150
8 30 000 7.5 ~ 500
16 9 – 12 000 2.25 – 3.0 ~ 150 - 200
Dilution experiments with mutation positive and wild-type samples showed ~ 60 fold coverage is required for mutation detection (including indels)
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NimbleGen Capture Array
Long oligonucleotide bound to chip
Exon 2Exon 1 Exon 4Exon 3 Exon 5
Fragment DNA and hybridise to NimbleGen capture array
Captured DNA fragments
Elute DNA and sequence using FLX
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NimbleGen Capture Array
Up to 5 MB of sequence can be captured
16 genes involved in muscular dystrophies defined – total sequence ~ 250, 000 base pairs
Projected capacity – 4 patients per LR70 sequenced for 16 genes
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NimbleGen Capture ArrayGene Chromosomal Location Total Size Captured sequence (to include exons)
LMNA 1q21.2-q21.3 25,410 8,015
FKRP 19q13.32 12,438 5,300
CAPN3 15q15.1-q21.1 64,214 31,600
CAV3 3p25 13,198 2,347
VCP 9p13.3 23,452 12,200
SGCA 17q21 9,926 6,400
SGCB 4q12 17,574 7,600
SGCD 5q33-q34 439,278 15,000
SGCG 13q12 144,213 5,250
DYSF 2p13.3-p13.1 233,130 63,300
LDB3 (ZASP) 10q22.3-q23.2 67,452 13,400
MYOT 5q31 19,979 9,300
DES 2q35 8,360 8,900
CRYAB 11q22.3-q23.1 3,144 3,700
FLNC 7q32-q35 28,853 25,300
DYSTROPHIN Xp21 2,092,328 29,647
TOTAL size (bp) 3,202,949 247,259
Capture 3 MB; capacity plate ~ 100 MB
max coverage = 33 fold (100/3) 1 patient per plate
Capture 250 kB; capacity plate ~ 100 MB
max coverage = 400 fold
process more than 1 patient per plate
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NimbleGen Capture Array
Processed one sample on LR70 PTP
NimbleGen Array
0.0
5.0
10.0
15.0
20.0
25.0
30.0
DYSFDM
D
CAPN3
FLNC
SGCDZASP
VCP
MYOT
LMNA
DES
SGCB
SGCA
SGCGFKRP
CRYABCAV3
Gene
Pe
rce
nta
ge
% of sequence on array
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NimbleGen Capture Array
113 MB sequence - ~ 50 % mapped to targets
NimbleGen Array
0.0
5.0
10.0
15.0
20.0
25.0
30.0
DYSFDM
D
CAPN3
FLNC
SGCDZASP
VCP
MYO
T
LMNA
DES
SGCB
SGCA
SGCGFKRP
CRYAB
CAV3
Gene
Pe
rce
nta
ge
% of sequence on array
% of seq mapping to gene
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Costs
Capture Array cost ~ £1000 Sequencing ~ £2500 (LR70 PTP) Cost for 16 genes = ~ £3500 processing 1 patient
per plate Conventional Sequencing = ~ £2500 for 1 patient Reduce costs by “tagging” patients prior to
processing on NimbleGen capture array Application for disorders with overlapping
phenotypes and multiple candidate genes (e.g. heart disease)
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Acknowledgements
Dr Jonathan Coxhead Dr Ann Curtis Dr Emma Ashton (Guy’s Hospital,
London)