Identification and monitoring of cancer mutations from cell free DNA-Seq data is a key application in liquid biopsy. In this part of the webinar we will show how mutations can be best identified from this type of data and how they can be interpreted. Furthermore, potential challenges when analyzing this type of data will be discussed together with relevant strategies.

1. Sample to Insight
Analysis and interpretation of cell-free DNA
Anika Joecker, Ph. D.
Director Global Product Management, Clinical Program, QIAGEN Bioinformatics
1Analysisand interpretation of cell-freeDNA

2. Sample to Insight
Legal Disclaimer
2
▪ QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention, or treatment of a disease.
▪ For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
Analysisand interpretation of cell-freeDNA

3. Sample to Insight
Agenda
3
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

4. Sample to Insight
Agenda
4
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

5. Sample to Insight
Why is cell-free DNA interesting?
• Applications
Identification of tumoral heterogeneity
Tracking of the evolution of the tumor genome over time
Identification of tumor recurrence and upcoming resistance, which leads to a change
in treatment
Non-invasive prenatal testing
5
• Advantages:
◦ Avoid surgeries and be able to take samples when no surgery is possible
◦ Achieve accuracy in the detection of all pathogenic and actionable variants
Analysisand interpretation of cell-freeDNA

6. Sample to Insight
Agenda
6
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

7. Sample to Insight
Challenges when analyzing cell-free DNA data from patients with cancer
7
 Due to dilution from non-cancerous cells, variants are expected at allele percentages of as low as 0.01%
 Most variant calling pipelines fail to call variants at this low percentage, which leads to many false negatives
 At this level the separation between true variants and false positives is very challenging, so comparisons are needed
 Changes in coverage and allelic dropout makes it challenging to compare samples
 Which variants are the ones driving the disease or are important for treatment decisions?
Analysisand interpretation of cell-freeDNA

8. Sample to Insight
Challenges when analyzing cell-free DNA data from patients with cancer
8
Problems when variant calling done with softwareA is not well connected with software B, which
is used for variant interpretation and software C, which is used for visualization
• Variant format available as export from software Ais not supported in software B
• Information gets lost between software Aand B
• Manual process: Variants have to be manually exported from software Aand imported into software B
and C.
A lot of time and
resources have to be
invested in building up
the system,
maintaining and
running it.
Software A
(Variant
calling)
Software B
(Data
interpretation )
Software C
(Result
visualization
for validation)
$
Analysisand interpretation of cell-freeDNA

9. Sample to Insight
Agenda
9
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

10. Sample to Insight
Sample to insight workflow
Title, Location,Date 10
Preparation
Collection
&
stabilization
Detection
Data analysis
&
Interpretation
• PAXgene Blood
ccfDNA Tubes
• QIAamp Circulating
Nucleic Acid Kit
• QIAsymphony
Circulating Nucleic
Acid Kit
• QIAseq cfDNA All-in-
One Kit
• QIAseq 1-Step
Amplicon Library Kit
• QIAseq Targeted DNA
Panels
• QIAseq FX DNA
Library Kit
• QIAseq Ultra Low
Input Library Kit
• Biomedical Genomics
Workbench
• Biomedical Genomics
Server Solution
• Ingenuity Variant
Analysis

11. Sample to Insight
Agenda
11
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

12. Sample to Insight
>30,000
Publications
16+ years
in bioinformatics
and literature
curation
>13,000,000
Biological findings:
• Disease relevant mutations
• Gene/Mutation – Drug
• Gene - Phenotype
• Gene – pathways relationships
Powered by
QIAGEN Bioinformaticssolutions are proven to scale
12Analysisand interpretation of cell-freeDNA

13. Sample to Insight
BiomedicalGenomics Workbench – complex tasks, simply done
13
Streamlined workflows and a rich toolbox to efficiently process data
Customize
workflows
QC reports
History
Visualization
and
Validation
Analysisand interpretation of cell-freeDNA

14. Sample to Insight
Specific functionalityfor target amplicon data
Remove amplicon primers after alignment and remove primer-dimer artefacts
14Analysisand interpretation of cell-freeDNA

15. Sample to Insight
Variant calling with Biomedical Genomics Workbench
15
Very sensitive and specific calling of variants at a very low level of 5% and even under 1%
Identification of known pathogenic mutations
directly from mapped sequencing reads
Accuracy for calling 5% low-frequency variants
using the default variant calling pipeline in
Biomedical Genomics Workbench
Analysisand interpretation of cell-freeDNA

16. Sample to Insight
Visualization of results
16
Visualization of causal variants in Ingenuity Variant Analysis
Analysisand interpretation of cell-freeDNA

17. Sample to Insight
Interpret and understand variants
Details panel provides links to findings, viewers and assessment
17Analysisand interpretation of cell-freeDNA

18. Sample to Insight
Pathway interpretation of variants and genes
Visualise variants on over-represented pathways, effects on genes and drugs that may
directionally target and effect pathway
18Analysisand interpretation of cell-freeDNA

19. Sample to Insight
Agenda
19
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

20. Sample to Insight
Case story: Comparison between primary tumor and cell-free DNA
20Analysisand interpretation of cell-freeDNA

21. Sample to Insight
Case story
21
Comparison between primary tumor and cell-free DNA
Analysisand interpretation of cell-freeDNA

22. Sample to Insight
Primary tumor (FFPE) and
buffy coat
FFPE: QIAamp DNA FFPE Tissue Kit
DNA from buffy coat: QIAamp DNA
Blood Mini Kit
Agilent SureSelectXT Human All Exon
V4 + UTR
HiSeq 2000
Biomedical Genomics Workbench
Ingenuity Variant Analysis
cfDNA
QIAamp Circulating Nucleic Acid Kit
Agilent SureSelectXT Human All Exon
V4 + UTR
HiSeq 2000
Biomedical Genomics Workbench
Ingenuity Variant Analysis
Sample
extraction
Enrichment
NGS
Data Analysis
Comparison between primary tumor and cell-free DNA
Data
Interpretation
22Analysisand interpretation of cell-freeDNA

23. Sample to Insight
Analysisand interpretation of cell-freeDNA 23
Step 1: Identification of somatic variants in primary tumor and cfDNA
Comparison between primary tumor and cell-free DNA
• Run on primary tumor, buffy coat
and cfDNA
• Conducts mapping of sequencing
reads and low frequency variant
calling
• cfDNA sample has to be run with a
frequency cutoff of 1% or under and
a read count of 5 reads at least
supporting the variant
• Sends results from all three samples
automatically to Ingenuity Variant
Analysis, filters results and returns
annotated and filtered variant list to
Biomedical Genomics Workbench to
be visualized in Genome Browser
• Variant Calling Results from primery
tumor and cfDNA has to be stated
as case and buffy coat as control

24. Sample to Insight
24
Step 1: Identification of somatic variants in primary tumor and cfDNA
Comparison between primary tumor and cell-free DNA
 Run on primary tumor, buffy coat and cfDNA
 Conducts mapping of sequencing reads and low
frequency variant calling
 cfDNA sample has to be run with a frequency
cutoff of 1% or under and a read count of 5
reads at least supporting the variant
 Sends results from all 3 samples automatically to
Ingenuity Variant Analysis, filters results and returns
annotated and filtered variant list to Biomedical
Genomics Workbench to be visualized in Genome
Browser
 Variant Calling Results from primary tumor and cfDNA
has to be stated as case and buffy coat as control
Analysisand interpretation of cell-freeDNA

25. Sample to Insight
Comparison between primary tumor and cell-free DNA
Analysisand interpretation of cell-freeDNA 25
Step 2: Modification of the filter cascade in Ingenuity Variant Analysis
1. Drag and drop the result
from Ingenuity Variant
Analysis (IVA) in an open
Genome Browser
2. Open the filter cascade in
Ingenuity Variant Analysis
for modification
3. Change Filter Cascade in
Ingenuity Variant Analysis
4. Fetch newest results from
Ingenuity Variant Analysis
5. Drag and drop updated
results from Ingenuity
Variant Analysis in Genome
Browser and compare
results

26. Sample to Insight
Analysisand interpretation of cell-freeDNA 26
Identified cancer driving variants by Ingenuity Variant Analysis for validation in Genome Browser
Comparison between primary tumor and cell-free DNA
Identified actionable variant in ESR1, just identified in cfDNA

27. Sample to Insight
Analysisand interpretation of cell-freeDNA 27
Problem: Some variants are not called, but are clearly present in primary tumor and/or buffy
coat
Comparison between primary tumor and cell-free DNA
Variant was identified in cfDNA, but not called in primary tumor, even when it is present.
Reason was that only a low fraction of reads support this variant in the primary tumor

28. Sample to Insight
Analysisand interpretation of cell-freeDNA 28
So, we are checking back into the sequencing reads to achieve a higher sensitivity
Comparison between primary tumor and cell-free DNA
Variants identified
in buffy coat
Variants identified
in primary tumor
Variants identified
in cfDNA
Read
mapping
buffy coat
Read
mapping
primary
tumor
Read
mapping
cfDNA
Combinedvariantlist from all samples
Check back into the
read mappings of all
samples to see if
variants seen in one
sample are also
present in other
samples

29. Sample to Insight
Analysisand interpretation of cell-freeDNA 29
Now the ESR1 variant seems also to be present in the primary tumor, but supported by just one
read. True or false positive?
Comparison between primary tumor and cell-free DNA

30. Sample to Insight
Comparison between primary tumor and cell-free DNA
Analysisand interpretation of cell-freeDNA 30
Number of potential cancer driver variants detected in cfDNA, primary tumor and buffy coat
41 3426
▪ 114 cancer drivers were detected in buffy coat, primary tumor and cfDNA together
▪ 26 variants and affected genes were directly associated with breast cancer
Primary Tumor cfDNA

31. Sample to Insight
Comparison between primary tumor and cell-free DNA
Analysisand interpretation of cell-freeDNA 31
Inspection of affected pathways

32. Sample to Insight
Analysisand interpretation of cell-freeDNA 32
...and drugs
Comparison between primary tumor and cell-free DNA

33. Sample to Insight
Analysisand interpretation of cell-freeDNA 33
Not described in paper: Variant in SLC9A2 found in cfDNA
Comparison between primary tumor and cell-free DNA

34. Sample to Insight
Agenda
34
Why is cell-free DNA interesting?1
Challenges when analyzing cell-free DNA
data from patients with cancer
2
QIAGEN sample-to-insight offering for cell
free DNA analysis
3
QIAGEN Bioinformatics solutions4
Case story5
6 Summary and questions
Analysisand interpretation of cell-freeDNA

35. Sample to Insight
Summary
35
 Biomedical Genomics Workbench and Ingenuity Variant
Analysis as streamlined solutions enable the easy
comparison of variants in primary tumor and cfDNA as
well as their interpretation and validation
 In the paper, validated variants in PIK3CA , ESR1 as well
as RNF144B in the breast cancer patient could be easily
detected, interpreted and confirmed together with
mapped sequencing reads
 The actionable variant in ESR1 was also detected in
primary tumor, but only supported by one read. This could
indicate that the mutation was already in the tumor, but
only in a very small number of reads
 Additional candidates were identified in the cfDNA, which
need to be validated
Analysisand interpretation of cell-freeDNA

36. Sample to Insight
Thank you for attending
Any questions?
Contact QIAGEN webinars
qiawebinars@QIAGEN.com
And QIAGEN Bioinformatics
BioinformaticsSales@qiagen.com
More information about products
shown at:
https://www.qiagenbioinformatics.com/
https://www.qiagen.com/
36Analysisand interpretation of cell-freeDNA