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

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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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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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DYSFDM

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CAPN3

FLNC

SGCDZASP

VCP

MYOT

LMNA

DES

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SGCGFKRP

CRYABCAV3

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% of sequence on array

NimbleGen Capture Array

113 MB sequence - ~ 50 % mapped to targets

NimbleGen Array

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SGCGFKRP

CRYAB

CAV3

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rce

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% 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)