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Dr Mark Davis
April 2015 MDWA Symposium
The Arrival Of Next Generation Sequencing in Perth
Next Generation Sequencing for
Neuromuscular Disorders
February 2011:
Mark Davis
Neurogenetics Unit, Department of Diagnostic Genomics,
PathWest Laboratory Medicine WA
The Arrival Of Next Generation Sequencing in Perth
October 2012:
The Arrival Of Next Generation Sequencing in Perth
June 2014:
So it’s rapidly evolving.
The Arrival Of Next Generation Sequencing in Perth
June 2014:
The Arrival Of Next Generation Sequencing in Perth
June 2014:
But what is it?
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Dr Mark Davis
April 2015 MDWA Symposium
Traditional Sequencing
Traditional Sequencing
Traditional (Sanger) sequencing involves analysis of each
exon as an individual reaction.
Small genes – easy: ACTA1 (6 exons)
3’
5’
Large genes – hard: DMD (79 exons)
Many genes – impossible:
Target Enrichment
CAPN3 (24 exons)
DYSF (55 exons)
TTN (364 exons)
Target Amplification
Make emulsion by
mixing
Denaturation
Ideal microreactor containing one
bead, one strand of template DNA,
primers and PCR mix
Annealing
Repeat PCR for
30 cycle
Extension
Adapted from Andy Vierstraete 2012
Sequencing
Custom Gene Capture Panel
(Neuromuscular V1)
• Construction (Q3 2012)
• 254 neurogenetic disease genes taken from the
Neuromuscular Disorders disease gene table (2012
freeze).
• 59 cardiac disease specific genes.
• 23 additional at that time unpublished candidate disease
genes.
• 336 genes in total.
• Capture Stats
• 6772 exons.
• 2.8 million base pairs of target sequence.
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Dr Mark Davis
April 2015 MDWA Symposium
Custom Gene Capture Panel
(Neuromuscular V1)
Custom Gene Capture Panel
(Neuromuscular V1)
Capture based: 3ug of DNA.
95% of targets covered to 20x or greater means 5% of targets
have less than 20x - effectively not covered.
Sample prep time: 3 days.
Run time: 4 hours (16 samples).
Initial data processing: 1 day.
This equates to 140kb over 338 exons.
Initially: Average coverage of ~200x, with 88-93% to 20x.
Now: Average coverage of ~300x, with 93-97% to 20x.
Some genes have coverage of 100% of the coding region,
some have coverage of 50%.
~1600 - 1800 variants, 60-80 remaining after initial filtering.
Routine Sanger infill of all holes is not viable.
Variant analysis: 5 minutes – 6 months.
Custom Gene Capture Panel
FKRP gene: ~50% of the coding region not covered to 20x –
misses the common mutation.
Custom Gene Capture Panel - Results
Disease
Cases
Positives
Ataxia
15
5
33%
All Cardiac
83
23
28%
CMD
37
19
51%
Channelopathy
27
14
52%
CMS
9
3
33%
CMT
97
22
22%
DA/FADS
29
5
17%
DIM
10
4
40%
GSD/rhabdo
46
13
28%
HSP
97
36
37%
LGMD/MD
119
51
43%
MND/SMA
13
2
15%
Myopathy
127
37
29%
Other
June 2013 – March 2015
6
1
715
234
33%
Up to and including run 78
Genes in which mutations have been reported:
AARS
ABCC9
ACTA1(x8)
ACTC1
ACTN1
AGL
ALS2
ANO5 (x4)
ATL1 (x2)
B3GALNT2 (x2)
BAG3
BSCL2
CACNA1A (x2)
CAPN3 (x11)
CHRNE
CHRNG
CLCN1 (x6)
COL6A1 (x7)
COL6A2 (x5)
COL6A3 (x5)
CPT2 (x2)
CYP7B1 (x2)
DMD (x14)
DNM2
DSP
DYSF (x6)
EGR2 (x2)
FA2H
FGF14
FKRP (x5)
GBE1
GDAP1 (x2)
GJB1 (x2)
GMPPB (x3)
GNE (x2)
HSPB1
IGHMBP2 (x2)
KCNH2
KIF5A
LAMA2 (x4)
LDB3
LMNA (x8)
MFN2 (x2)
MPZ (x4)
MTM1 (x3)
MYBPC3 (x6)
MYH2
MYH3 (x2)
MYH7 (x3)
MYL3
MYOT (x2)
NEB (x5)
NIPA1 (x2)
OPA1
PLP1
POMGNT1
POMT1 (x4)
POMT2 (x2)
PRX
PYGM (x2)
RAPSN (x2)
RMB20
REEP1 (x3)
RYR1 (x12)
SACS (x5)
SCN4A (x8)
SCN5A (x2)
SEPN1 (x2)
SGCA (x4)
SGCB
SGCG
SH3TC2 (x3)
SOD1
SPAST (x7)
SPG7 (x18)
SPG11
TNNT2 (x2)
TNNT3
TPM2 (x3)
TRPV4 (x2)
TTN (x13)
WNK1
262 cases, 82 genes involved.
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Dr Mark Davis
April 2015 MDWA Symposium
Findings (LGMD/MD)
Genes in which mutations have been reported:
AARS
ABCC9
ACTA1(x8)
ACTC1
ACTN1
AGL
ALS2
ANO5 (x4)
ATL1 (x2)
B3GALNT2 (x2)
BAG3
BSCL2
CACNA1A (x2)
CAPN3 (x11)
CHRNE
CHRNG
CLCN1 (x6)
COL6A1 (x7)
COL6A2 (x5)
COL6A3 (x5)
CPT2 (x2)
CYP7B1 (x2)
DMD (x14)
DNM2
DSP
DYSF (x6)
EGR2 (x2)
FA2H
FGF14
FKRP (x5)
GBE1
GDAP1 (x2)
GJB1 (x2)
GMPPB (x3)
GNE (x2)
HSPB1
IGHMBP2 (x2)
KCNH2
KIF5A
LAMA2 (x4)
LDB3
LMNA (x8)
MFN2 (x2)
MPZ (x4)
MTM1 (x3)
MYBPC3 (x6)
MYH2
MYH3 (x2)
MYH7 (x3)
MYL3
MYOT (x2)
NEB (x5)
NIPA1 (x2)
OPA1
PLP1
POMGNT1
POMT1 (x4)
POMT2 (x2)
PRX
PYGM (x2)
RAPSN (x2)
RMB20
REEP1 (x3)
RYR1 (x12)
SACS (x5)
SCN4A (x8)
SCN5A (x2)
SEPN1 (x2)
SGCA (x4)
SGCB
SGCG
SH3TC2 (x3)
SOD1
SPAST (x7)
SPG7 (x18)
SPG11
TNNT2 (x2)
TNNT3
TPM2 (x3)
TRPV4 (x2)
TTN (x13)
WNK1
LGMD/MD (51/119):
ACTA1
ANO5 (x3)
CAPN3 (x10)
COL6A1
COL6A2
DMD (x13)
DYSF (x4)
FKRP (x6)
LAMA2
LMNA (x3)
MYOT
POMT1
SGCA (x3)
SGCB
SGCD
TPM2
262 cases, 82 genes involved – 135 cases are in 52 ‘new’ genes.
Findings (LGMD/MD)
LGMD/MD (51/119):
ACTA1
ANO5 (x3)
CAPN3 (x10)
COL6A1
COL6A2
DMD (x13)
DYSF (x4)
FKRP (x6)
LAMA2
LMNA (x3)
MYOT
POMT1
SGCA (x3)
SGCB
SGCD
TPM2
The study highlighted a lack of relationship between dystrophic
phenotype and X-inactivation pattern in females.
Issues

Gaps in coverage:
• Repeats.
• Expansions.
• Large deletions/insertions.
• High GC content.
 Data interpretation:
• Sequencing errors (particularly homopolymer runs).
• Many variants of uncertain significance.
Issues

Gaps in coverage:
• Repeats.
• Expansions.
• Large deletions/insertions.
• High GC content.

Data interpretation:
• Sequencing errors (particularly homopolymer runs).
• Many variants of uncertain significance.
We need as much clinical information as possible (including results
of any conventional testing) to narrow the list down and allow
meaningful analysis.
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Dr Mark Davis
April 2015 MDWA Symposium
Issues

Summary
Gaps in coverage:
• Repeats.
• Expansions.
• Large deletions/insertions.
• High GC content.
• The Neuromuscular NGS panel gives a much improved pickup
rate at lower cost in less time.

Data interpretation:
• Sequencing errors (particularly homopolymer runs).
• Many variants of uncertain significance.
• Reiteration of the panel to include new genes has just arrived –
panels have a life for a few more years.

Knowledge base:
• Switch to WES.
•

Reporting on genes we have not previously studied
Data storage:
•
Each patient file is ~ 6 GB (largest hospital network drive is
• Whole genome sequencing (which will cover CNVs and include
most cytogenetics).
1.8TB).
Acknowledgements
The Future - Whole Genome Sequencing?
NGS Facility
Neurogenetics
Harry Perkins
Richard Allcock
Nina Kresoje
Kyle Yau
Vanessa Atkinson
Cheryl Wise
Rebecca Gooding
Padma Sivadorai
Fathimath Faiz
Dan Trajanoski
Mei Ting Chiew
William Wallefeld
Nigel Laing
Gina Ravenscroft
Macarena Cabrera
Royston Ong
Sarah Beecroft
Not yet……
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