ngs, a suitable approach for tp53 screening in cll? - eric · ngs, a suitable approach for tp53...
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NGS, a suitable approach forTP53 screening in CLL?
Ferran Nadeu
2nd ERIC WORKSHOP ON TP53 ANALYSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA7-8 November 2017, Stresa (Italy)
1 patient
TP53
4 PCR/case*
+
* Exon 4, exons 5-6, exon 7, exons 8-9.
=
4 PCRs
20 patients
TP53
4 PCR/case*
+
=
100 PCRs
400 patients
TP53
4 PCR/case*
+
=
1600 PCRs
The Sanger sequencing bottleneck
1 patient
TP53
4 PCR/case*
+
=
4 PCRs
20 patients
TP53
4 PCR/case*
+
=
100 PCRs
400 patients
TP53
4 PCR/case*
+
=
1600 PCRs
TP53, SF3B1, BIRC3,
NOTCH1 and ATM
≈75 PCR/case
75 PCRs 1500 PCRs
TP53, SF3B1, BIRC3,
NOTCH1 and ATM
≈75 PCR/case
30000 PCRs
TP53, SF3B1, BIRC3,
NOTCH1 and ATM
≈75 PCR/case
The Sanger sequencing bottleneck
Nadeu et al Blood 2016
Outline
1. NGS overcomes this Sanger sequencing bottleneck (focus on the Access-Array system)
2. NGS for TP53 analysis:
a) Access-Array system
b) Comparison of NGS libraries
3. Pros and cons of the use of NGS:
a) Sensitivity
b) Bioinformatic analysis
c) Accuracy
d) Possibility to study several genes simultaneously (i.e. panel of genes)
Access-Array system (Fluidigm) – Quick overview
Amplicon-based
15 amplicons of 180bp
4-Primer Amplicon Tagging
Access-Array system (Fluidigm) – Quick overview
48.48 Access Array IFC (Integrated Fluidic Circuit)
IFC controller AX
2304 individual reactions
chambers (30 nL)
Access-Array system (Fluidigm) – Quick overview
48.48 Access Array IFC (Integrated Fluidic Circuit)
IFC controller AX BioMark HD
Access-Array system (Fluidigm) – Quick overview
48.48 Access Array IFC (Integrated Fluidic Circuit)
IFC controller AX
Access-Array system (Fluidigm) – Quick overview
48.48 Access Array IFC (Integrated Fluidic Circuit)
Clean-up
Access-Array system – Up to 480 amplicons
Multiplexing
Figures adapted from Access-Array System User Guide (Fluidigm).
2nd PCR done in a
96-well plate in a
or al ther ocycler
Outline
1. NGS overcomes this Sanger sequencing bottleneck (Access-Array system)
2. NGS for TP53 analysis:
a) Access-Array system
b) Comparison of NGS libraries
3. Pros and cons of the use of NGS:
a) Sensitivity
b) Bioinformatic analysis
c) Accuracy
d) Possibility to study several genes simultaneously
Coverage/depth in NGS
A T G C C T G A T G
Spencer et al J Mol Diagn 20142 4 6 6 6 6 6 4 2 0Coverage
T G C C T G A
A T G C C T G
G C C T G A T
T G C C T G A
G C C T G A T
A T G C C T G
Access-Array system (Fluidigm) – Coverage
Nadeu et al Blood 2016
A coverage >1000x was obtained in >85% of the sequence in 95% of the samples
Capture hybridization-based
Amplicon-based
• Acces-Array - Flluidigm
• AmpliSeq - ThermoFisher (Ion Torrent)
• Genereadv2/QIASeq - QIAgen
•Multiplicom - Agilent
• TruSeq Custom Amplicon - Agilent
• TruSeq Custom Amplicon DS – Agilent
• NexteraXT - Illumina
•SureSelect - Agilent
• SeqCap EZ System -Nimblegen/Roche
• xGen Lockdown - IDT
• Haloplex – Agilent
• Nextera XT - Illumina
Comparison of NGS libraries for TP53 screening
Outline
1. NGS overcomes this Sanger sequencing bottleneck (Access-Array system)
2. NGS for TP53 analysis:
a) Access-Array system
b) Comparison of NGS libraries
3. Pros and cons of the use of NGS:
a) Sensitivity
b) Bioinformatic analysis
c) Accuracy
d) Possibility to study several genes simultaneously
Pros and cons of NGS: Sensitivity
Rossi et al Blood 2014:
• 30% (15/50) patients carried only subclonal TP53 mutations
• 8% (4/50) patients carried isolated del(17p)
• NGS: 92% (46/50) of patients with TP53 alterations
• Sanger + FISH: 70% (35/50) of patients with TP53 alterations
Nadeu et al Blood 2016:
• 34% (16/47) patients carried only subclonal TP53 mutations
• 9% (4/47) patients carried isolated del(17p)
• NGS: 91% (43/47) of patients with TP53 alterations
• Sanger + SNP array: 66% (31/47) of patients with TP53 alterations
Pros and cons of NGS: Bioinformatic analysis
Sequencing error rate:
Platform
Chemistry
Read length
Genomic context
...
Spencer et al J Mol Diagn 2014
Pros and cons of NGS: Accuracy
Nadeu et al Blood 2016
VAF ≥
%
VA
F <
12
%
(Sanger sequencing on
302 regions/genes detected 69 mutations)
100% specificity and
100% sensitivity
(43 cases carrying high frequency mutations
were subjected to a second round of NGS)
100% reproducibility
88 mutations verified by AS-PCR
77 mutations verified by a 2nd round of NGS
13/13 were also verified by AS-PCR
Verification: AS-PCR or 2nd run of NGS
After a non-stringent custom filtering ≈75% specificity < % of VAF
Pros and cons of NGS: Scalability
Genes targeted
Cost
Hands-on
Precision medicine
1 nNumber of genes targeted
n
0
Tim
eE
uro
sB
en
efi
t
NGS libraries and TP53
• Easy design
• Short hands-on time
• Compatible with multiple sequencing platforms (Illumina, Ion Torrent, etc.)
• Percentage of amplification success ≥95%, and good accuracy (>90% mapping to target)
• Uniform coverage
• Highly sensitive and reproducible approach to detect mutations in TP53 and other genes in clinical samples
Take home messages
NGS in the clinics
• Many different library approaches work well.
• We may face a CLL (or lymphoma) panel rather than analyzing an individual gene
• Which genes? How we validate it in larger cohorts?
• Should we sequence the normal DNA? It could be useful for the study of ATM mutations and for the variant calling.
Bioinformatic approach (still not well defined)
• A gold-standard bioinformatic pipeline for deep-targeted NGS is not yet stablished.
• Limited accuracy in the detection of clinically relevant very low VAF mutations. Verification may be needed.
• Feasible identification of CNA [del(17p), del(11q)] from the same NGS data is still pending.
…should all this be uniform for the different centers?