The Challenges Of Sequencing FFPE DNA Using NGS Hazel Ingram

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.

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

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

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

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

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)

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.

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.

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.

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

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

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.

Acknowledgements Alex Stretton James Eden Helen Roberts Rachel Butler Matt Mort (HGMD) The All Wales Medical Genetics Service Hazel.ingram@wales.nhs.uk