1. The Challenges Of Sequencing FFPE DNA Using NGS
Hazel Ingram

2. Problems with FFPE Poor quality/fragmented DNA Fixation artefacts
Insufficient tumour material
Low tumour content
Measuring Quality
Nanodrop vs Qubit
Fragmentation
I’m sure anyone who has worked with FFPE samples before knows how difficult they are to work with. But they are the most widely practiced method for clinical sample preservation and archiving.
It is often poor quality/fragmented DNA  due to the fixation process – fragmented and cross-linked DNA, with high conc of contaminants, the number of amplifiable copies of DNA are reduced by strand breaks and other DNA damage.
A common problem is fixation artefacts, formalin causes C>U deamination which can appear like a genuine C>T mutation – false positives
This isn’t always the case, but often we can have Insufficient tumour material – particularly if the sample is a biopsy
In addition to that the sample could have a low tumour content  we need to think about the sensitivity of our tests.
To try and help with this we macrodissect, we ask a histopathologist to mark on an H&E slide where the tumour cells are found, and what percentage of tumour cells are found in that area.
Measuring quality is also important, we use the Qubit to measure concentration for NGS work, as it is much more accurate than the Nanodrop. It would also be useful to measure the fragmentation.

3. Projects CRUK SMP1 FOCUS 4 WCB Sample Duplicate Single Library Prep
CRUK SMP1
FOCUS 4
WCB
Sample
Duplicate
Single
Library Prep
TSCA
Haloplex
Sequencing
MiSeq
Analysis
MiSeq Reporter
NextGENe
HGMD – custom pipeline
Thresholds for variant calling
5% sensitivity
150x coverage per replicate
150x coverage
50x coverage
We currently run 3 research based studies using FFPE tissue for a variety of different tumour types
CRUK SMP (KRAS, PIK3CA, BRAF, NRAS, TP53, PTEN, EGFR, DDR2) –I’ll be talking mainly about this project, as it’s what I’ve been working on.
FOCUS4 (KRAS, PIK3CA, BRAF, NRAS, PIK3R1)
WCB (KRAS, PIK3CA, BRAF, NRAS, TP53, PTEN)
Each of these 3 research projects involves investigation of a similar panel of genes, including KRAS, NRAS and BRAF.
Initially, pyro and Sanger sequencing was used and NGS was implemented to streamline the testing and analysis process

4. Validation Determine acceptable coverage for regions of interest
To match or increase sensitivity compared to original methods (pyro and sanger)
Low level of false positives
Must have at least 90% concordance
The validation process is the same for all new panels and involves testing the panel with samples that have known mutations.
We need to detemine what is an acceptable level of coverage for the ROI
To at least match the sensitivity of the test compared to the original methods
We need to check we are maintaining a low level of false positives
And we must have at least 90% concordance

5. CRUK SMP1 Validation Overview
Illumina validation:
42 samples sent:
37/42 matched (88%)
3/42 non concordant
low coverage
2/42 non concordant
design issue
In House validation:
45 samples
43 matched (95%)
2/45
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
Low Coverage
Technological Design
Higher Sensitivity of NGS
So just a little bit of background on CRUK Phase I SMP trial – there are 3 TH doing the lab work, and 9 CHs. Looked at a variety of tumour types and genes. The NGS panel was designed by Illumina, so we were obliged to use their TSCA library prep kit
1st part of validation was sending samples to Illumina to test their panel, a total of 42 samples were sent, each with at least one mutation– 37/42 (88%), of the 5 that were non concordant, 3 of those missed mutations were due to low coverage, 2 of these were due to design, so the panel was tweaked, and we moved onto the second part of validation
2nd part of the validation, was in house, and this is the sort of validation that was done for the WCB and FOCUS-4. So we tested 45 old samples, and had 95% concordance, again the 2 that didn’t match were due to low coverage. Between the 3 THs, in house 124 samples were tested and 18 additional variants were detected – either through failure of original tech or higher sensitivity of NGS
It was also apparent that a large number of artefacts were being produced.
TSCA gave a similar failure rate to the original methods (pyro and sanger seq) of approx 10%, but because the TSCA panel extended outside of the original regions, suggesting overall failure rate is lower.
The important points here are:
There were lots of artefacts
low NGS coverage means missed mutations
Technological design
We proved that there NGS had higher sensitivity

6. Artefacts  Duplicate Testing
True variants seen in both replicates
Pipeline - only sequence variants observed in both replicates retained for analysis
Solution = testing of each sample in duplicate
Sequencing artefacts only seen in 1 replicate
Strategy
Average number of
variants per sample
Range in number of
Singlicate testing
113
3-546
Duplicate testing 30
0-60
When we were doing the CRUK validation, we soon noticed that there were a very large number of artefacts per sample. This was a big problem, as we had limited resources and timescale for analysing and interpreting this volume of data. We moved towards duplicate testing, and discovered on most occasions, seq artefacts were only seen in 1 variant and true variants seen in replicates, we used our pipeline to filter so that only those seen in both replicates were retained for analysis
* Duplication used for CRUK panel only

7. Artefacts - Sanger Seq False Positive (deamination)
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
Just as another point to mention, one of the non-concordant results from CRUK validaton was found to be the result of a false positive in Sanger sequencing, rather than a false negative in NGS analysis.
-In this case, a melanoma FFPE sample was identified as having a low level nonsense mutation in the KIT gene by Sanger, as shown in the trace.
-But no KIT mutation was identified by NGS. Re-analysis by Sanger sequencing was performed and showed WT sequence.
Therefore this ‘mutation’ was concluded as being a C>T deamination artefact caused by formalin fixation of the FFPE sample.
Steps can be taken to help these sort of artefacts, and there have been papers on the effect of UDG treatment prior to PCR which may help. (UDG is a DNA repair enzyme that removes the uracil lesions by hydrolysing N-glycosidic bond)

8. Minimum Coverage: CRUK & FOCUS4
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
There needs to be a minimum coverage for QC purposes.
Looking at our validation data known variants became undetectable when coverage for ROI dropped below 150x, so this is much higher than we would say for Germline – but we are working with much smaller percentage mutant.
So for CRUK and FOCUS4 150x minimum coverage cut off was implemented, for CRUK, if there was less than 150x coverage in either replicate the exon was failed. This meant we avoided reporting false negative results, and maintained reasonable failure rates comparable to original testing.

9. Design CRUK (TSCA) FOCUS4 (TSCA) WCB (Haloplex) 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
I’ll just give an example of a design issue in each of the panels
CRUK – looked into this, there was good coverage (>4000 reads), discovered that the deletion removed the probe binding site for the 2 probes in this region, so there was a redesign and a different probe set was included in the panel
FOCUS 4 – missed this NRAS mutation, when we investigated we noticed that the region has no reads for this patients and half of the other validaiton patients had no coverage for the same region, so did an assay redesign to get better amplification.
WCB – used a different library prep method, which involves an enzyme digest. again a redeisgn was necessary… these are just some of the reasons why design and validation is very important.

10. Library Prep Methods: TSCA vs Haloplex
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
100%
TSCA - NextGENe 26
93%
For some projects we were restricted by what library prep to use, but for the WCB project we have a lovely lady Alex who researched into different panels. A comparison was done, by our researcher Alex, into TSCA and haloplex.
Both panels were designed to cover the same regions of interest- which included KRAS, BRAF, NNRAS, PIK3CA, and samples were set up and then run on the MiSeq, both were analysed with NextGene software for a direct comparison
So as you can see the haloplex performed slightly better to the TSCA, there were 2 missed mutations.

11. TSCA vs Haloplex
As another comparison, some of you may be familiar with IGV- a viewing software, so this is the same region for the same patient, all of the greys indicate matching bases to the ref seq and the coloured lines represent mis-matches. The top picture shows Haloplex data, and the bottom TSCA, so much more artefacts in this run. So, haloplex kit was picked for use with the WCB project

12. Projects CRUK SMP FOCUS 4 WCB Sample Duplicate Single Library Prep
CRUK SMP
FOCUS 4
WCB
Sample
Duplicate
Single
Library Prep
TSCA
Haloplex
Sequencing
MiSeq
Analysis
MiSeq Reporter
NextGENe
HGMD – custom pipeline
Thresholds for variant calling
5% sensitivity
150x coverage per duplicate
150x coverage
50x coverage
And it was partly down to this cleaner data that the minimum coverage level was put much lower than the other projects

13. Conclusions and Future Work
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
So the DNA quality from FFPE tissue is a major challenge, but it is unlikely that we will move from this method of preservation in the near future, because it maintains tissue morphology allowing for macrodissection, and is easy and cheap to store in comparison to fresh frozen tissue.
we’ve discovered that running in duplicate and being careful with assay design and minimum coverage cut off helps with the interpretation of these samples.
Adding steps, e.g. UDG treatment prior to PCR step can help minimise deamination artefacts
For the future, we need a more robust NGS technology for low quality DNA from FFPE – that could be different NGS platforms or different enrichment methods.

14. Acknowledgements Alex Stretton James Eden Helen Roberts Rachel Butler
Matt Mort (HGMD)
The All Wales Medical Genetics Service