The Challenges Of
Sequencing FFPE DNA
Using NGS
Hazel Ingram
Problems with FFPE
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Poor quality/fragmented DNA
Fixation artefacts
Insufficient tumour material
Low tumour content
Measuring Quality
• Nanodrop vs Qubit
• Fragmentation
Projects
CRUK SMP1
FOCUS 4
WCB
Sample
Duplicate
Single
Single
Library Prep
TSCA
TSCA
Haloplex
Sequencing
MiSeq
MiSeq
MiSeq
Analysis
MiSeq Reporter
NextGENe
NextGENe
5% sensitivity
5% sensitivity
150x coverage
50x coverage
HGMD – custom
pipeline
Thresholds for 5% sensitivity
variant
150x coverage per
calling
replicate
Validation
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Determine acceptable coverage for regions
of interest
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To match or increase sensitivity compared
to original methods (pyro and sanger)
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Low level of false positives
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Must have at least 90% concordance
CRUK SMP1 Validation Overview
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Illumina validation:
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42 samples sent:
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In House validation:
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45 samples
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37/42 matched (88%)
3/42 non concordant
• low coverage
2/42 non concordant
• design issue
Artefacts
Low Coverage
Technological Design
Higher Sensitivity of NGS
43 matched (95%)
2/45
• low coverage
Overall, between hubs 18 extra variants previously undetected in 124 samples
• Technological fail of pyro/sanger
• Higher sensitivity of NGS
Large number of artefacts detected
Failure rate: approx 10%
Artefacts  Duplicate Testing
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Solution = testing of each
sample in duplicate
Sequencing artefacts only
seen in 1 replicate

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True variants seen in both
replicates
Pipeline - only sequence
variants observed in both
replicates retained for analysis
Strategy
Average number of
variants per sample
Range in number of
variants per sample
Singlicate testing
113
3-546
Duplicate testing
30
0-60
* Duplication used for CRUK panel only
Artefacts - Sanger Seq False Positive (deamination)
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KIT c.1745G>A
p.Trp582X – CRUK SMP
Mutation not detected
by NGS (single testing:
>4000x)
Mean coverage across
panel: 4840x
KIT re-tested by
Sanger…no mutation
detected
C>T deamination
artefact caused by
formalin fixation
…duplicate testing
solves this issue
UDG treatment prior to PCR may help
 removes uracil lesions by
hydrolysing N-glycosidic bond
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Minimum Coverage: CRUK & FOCUS4
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Known variants became undetectable when
coverage for region of interest <150x
150x minimum coverage cut-off implemented
• For CRUK: <150x coverage in either replicate = failed
exon
• Avoided reporting false negative results
• Failure rates were still comparable to original
testing
Design
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CRUK (TSCA)
•
PTEN 27bp del missed
• Deletion removed probe binding site
FOCUS4 (TSCA)
•
NRAS c.182A>G p.Q61R
• Region had zero reads  poor amplification
WCB (Haloplex)
•
PIK3CA ex9 c.1634A>G, p.E545G missed
Library Prep Methods: TSCA vs Haloplex
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Both panels designed to cover same ROI
Same samples run on both panels
Run on MiSeq and analysed using NextGENe for
direct comparison
Number of
samples
Number of
expected
mutations
Number of
mutations
found
Percentage
Haloplex –
NextGENe
11
28
28
100%
TSCA NextGENe
11
28
26
93%
TSCA vs Haloplex
Projects
CRUK SMP
FOCUS 4
WCB
Sample
Duplicate
Single
Single
Library Prep
TSCA
TSCA
Haloplex
Sequencing
MiSeq
MiSeq
MiSeq
Analysis
MiSeq Reporter
NextGENe
NextGENe
5% sensitivity
5% sensitivity
150x coverage
50x coverage
HGMD – custom
pipeline
Thresholds for 5% sensitivity
variant
150x coverage per
calling
duplicate
Conclusions and Future Work
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DNA quality from FFPE tissue is a major challenge
Help interpretation by:
•
•
Running in duplicate
being careful with assay design and minimum coverage
Additional steps e.g. UDG treatment can help
minimise deamination artefacts
Need a more robust NGS technology for low
quality DNA from FFPE
• Alternative NGS platforms / providers
• Alternative enrichment methods e.g. target capture as
alternative to PCR based
Acknowledgements

Alex Stretton
James Eden
Helen Roberts
Rachel Butler
Matt Mort (HGMD)
The All Wales Medical Genetics Service

Hazel.ingram@wales.nhs.uk
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