Early experiences in amplicon sequencing using the Roche GS-FLX massively parallel DNA sequencer and...
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Transcript of Early experiences in amplicon sequencing using the Roche GS-FLX massively parallel DNA sequencer and...
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
GS-FLX - Roche
Next Generation DNA Sequencer
GS-FLX - Roche
Next Generation DNA Sequencer Based upon pyrosequencing
GS-FLX - Roche
Next Generation DNA Sequencer Based upon pyrosequencing
Sequencing Reagents
Camera
Work-flow
Library preparation Emulsion PCR – Clonal Amplification Sequencing Data analysis
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
Emulsion PCR
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Binding
Emulsion Formation
PCR Amplification
Emulsion PCR
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Binding
Emulsion Formation
PCR Amplification
Emulsion PCR
+
Binding
Emulsion Formation
PCR Amplification
Emulsion PCR
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Binding
Emulsion Formation
PCR Amplification
Emulsion PCR
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Binding
Emulsion Formation
Emulsion Breaking and Bead Enrichment
Sequencing
Occurs on PicoTitre Plates Capacity of LR70 ~ 100 MB Split into 2, 4, 8 or 16 regions
Sequencing
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Sequencing
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Sequencing
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Sequencing
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Sequencing
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Sequencing
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Sequencing
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G T A A G T T T T
Sequencing
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G T A A G T T T T C C C
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
Data Interpretation
Length of amplicon sequence
Number of reads across amplicon
Variants
Variant Table
Consensus Global Alignment Plot
Data InterpretationVariant Table
Consensus Global Alignment Plot
T>CC>T
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
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]
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)
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
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
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
NimbleGen Capture Array
Processed one sample on LR70 PTP
NimbleGen Array
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5.0
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DYSFDM
D
CAPN3
FLNC
SGCDZASP
VCP
MYOT
LMNA
DES
SGCB
SGCA
SGCGFKRP
CRYABCAV3
Gene
Pe
rce
nta
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% of sequence on array
NimbleGen Capture Array
113 MB sequence - ~ 50 % mapped to targets
NimbleGen Array
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DYSFDM
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CAPN3
FLNC
SGCDZASP
VCP
MYO
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LMNA
DES
SGCB
SGCA
SGCGFKRP
CRYAB
CAV3
Gene
Pe
rce
nta
ge
% of sequence on array
% of seq mapping to gene
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)
Acknowledgements
Dr Jonathan Coxhead Dr Ann Curtis Dr Emma Ashton (Guy’s Hospital,
London)