Combining semiconductor-based sequencing with amplicon-based cancer
gene panels: A rapid next-gen approach to clinical cancer genotyping.
Knight Diagnostic Laboratories
Christopher L. Corless, Tanaya Neff, Michael C. Heinrich, Carol Beadling
Knight Cancer Institute, Oregon Health & Science University, and Portland Veteran's Affairs Medical Center, Portland, Oregon
AmpliSeq Cancer Gene Panel (Ion Torrent)
Background: Bringing next-gen sequencing into clinical (CLIAlicensed) laboratories is an important step in the advancement of
personalized cancer care. We have validated a new
semiconductor –based sequencing approach (Ion Torrent PGM)
in combination with amplicon-based library preparations. Using
FFPE-derived samples of tumor and normal DNA , the
performance of two panels has been examined in detail: 1) the
AmpliSeq Cancer Panel (available from Ion Torrent); 2) a custom
panel designed for genotyping GI stromal tumors (GISTs).
Genomic DNA
Amplify gDNA targets
190-Amplicon multiplex PCR
Ion AmpliSeqTM Cancer Primer Pool
Results
• On-target reads: 95%
• Average read length:76 bp
• Average read depth: 2000 (range 8 – 4,948)
• All 53 point mutations were identified.
• All 19 in/dels were visible in the sequence, but most were not
accurately identified by the Torrent Suite software (version 2.0).
•Using a cut-off of 8% mutant allele, the sensitivity for known
mutations was 100% and the specificity was 92%.
•There were 27 new non-synonymous mutations with >8% mutant
allele frequency detected in regions not covered by the mass
spectrometry-based panel, including 24 point mutations and 3
deletions of 2 bp; all 27 were confirmed by Sanger sequence
analysis.
MassArray Allele Ratio
50%
• 49.1 kb of target sequence
40%
KRAS
MAP2K1
NF1
NRAS
PDGFRA
PIK3CA
PTEN
PTPN11
SDHA
SDHAF1
SDHAF2
SDHB
SDHC
SDHD
TP53
30%
Results
• On-target reads: 88%
• Average read length: 86 bp
• Average read depth: 695 (range 11 – 1,848)
• An NRAS Q61L mutation was confirmed in one case; this
mutation is novel in GIST
• Barcoded samples
20%
BC
3000
P1
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
AmpliSeq Allele Ratio
Emulsion PCR
Enrichment of templated IonSphere particles
Sequence
2500
6000
Reads per amplicon
(Mean + standard dev.)
5000
Reads per amplicon
(Mean + standard dev.)
0%
Average number of reads
A
4000
2000
Conclusions:
1500
1000
3000
2000
500
1000
0
1
11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251 261 271 281 291 301 311 321 331 341 351 361 371
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
Amplicon
190
Amplicon
12
Number of targets within read depth
% of targets ≥ read depth
10
70
90%
80%
70%
8
60%
6
50%
40%
4
30%
20%
2
10%
0
0%
Read depth
100%
Number of targets within read depth
100%
Number of target regions with read depth
• 4 samples per 316 chip
• 125 to 175 bp amplicons
60%
Percentage of targets ≥ read depth
• Barcoded samples
Ligate barcode adapters
Nick-translate and amplify
R=0.92, P<0.0001
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
3100
3200
3300
3400
3500
3600
3700
3800
3900
4000
4100
4200
4300
4400
4500
4600
4700
4800
4900
5000
• 13.3 kb of target sequence
PDGFRA
PIK3CA
PTEN
PTPN11
RB1
RET
SMAD4
SMARCB1
SMO
SRC
STK11
TP53
VHL
• Amplicons tiled for
whole gene coverage
53 Point Mutations
70%
AKT1
AKT2
AKT3
ATM
BRAF
CDKN2A
HRAS
KIT
• Primers were designed with the assistance of collaborators at
Ion Torrent using the Ion AmpliSeq designer software v1.0.
• Samples tested: 19 FFPE GISTs that were previously shown to
be negative for KIT and PDGFRA mutations.
• 8 barcoded samples run on a single 318 chip, yielding an
average of 337,000 reads per sample
• 11 barcoded samples run on 316 chips (@ 4 per chip), yielding
an average of 392,000 reads per sample
10%
Number of target regions
• 190 primer pairs (1 tube)
FLT3
GNAS
HNF1A
HRAS
IDH1
JAK2
JAK3
KDR
KIT
KRAS
MET
MLH1
MPL
NOTCH1
NPM1
BC
P1
Average number of reads
• Input DNA: 10 ng
AKT1
ALK
APC
ATM
BRAF
CDH1
CDKN2A
CSF1R
CTNNB1
EGFR
ERBB2
ERBB4
FBXW7
FGFR1
FGFR2
Red= 1:1 equivalence
Remove genomic DNA template and primers
Partially digest primers
A
• 477 primer pairs (2 tubes)
90%
80%
AmpliSeq Cancer Gene Panel (Ion Torrent)
• Designed to detect hotspot mutations across 46 genes.
• Samples tested: 45 FFPE solid tumors previously genotyped on
Sequenom MassArray system, including :
- 53 point mutations
ABL1
FGFR3
NRAS
- 19 in/dels (4 – 63 bp)
Comparison of allele ratios:
MassArray vs AmpliSeq
100%
Custom GI Stromal Tumor (GIST) Panel
• Input DNA: 20 ng
90%
% of targets ≥ read depth
60
80%
50
70%
60%
40
Average 695 reads
50%
369/380 = 97% >0.2x mean
30
40%
30%
20
20%
10
10%
0
• Combining solid-state sequencing with a highly multiplexed
PCR method for library construction is a rapid (48 hr)
approach for next-gen sequencing of clinical cancer samples.
• The protocol requires very little DNA (10-20 ng). Indeed, two
DNA samples from laser-captured tumor yielded excellent
data with the AmpliSeq Cancer Panel.
• The process is highly scalable and larger, cancer-specific
amplicon panels are in development.
• Automated identification of in/dels remains a challenge in
next-gen sequencing output.
• Recent improvements in the library preparation protocol and
in the variant caller software are leading to further reductions
in turn-around time and the number of background variants.
• Both the Cancer Gene Panel and the GIST Panel are now
available in our CLIA-licensed/CAP-accredited laboratory.
0
100
200
300
400
500
600
700
800
900
1000
Read Depth
1100
1200
1300
1400
1500
1600
1700
1800
0%
Acknowledgements: We are grateful for the help of our
collaborators at Ion Torrent, including Katherine Rhodes,
Michael Thornton, John Leamon and Mark Andersen.