through this slide show we will explain the importance of clinical consideration when considering clinical reporting from an NGS based test

1. NGS Variant Calling: Clinical
Point of View
Mohamed Kamal Hussein
NGS Clinical Implementation Consultant
Founder and CEO Omicsense

2. Content
• NGS variant calling overview.
• Germline Mutation VS Somatic mutation.
• Clinical annotation databases and variant effect prediction.
• Clinical importance algorithm or variant prioritization.
• Sample flow in real NGS clinical lab.
• Take home Message
• Questions

3. NGS Variant Calling Overview
What is Variant Calling?
• Variant is the specific region of the genome which differs between
two genomes.
• Alleles are just the different versions of that Variant, so a SNP may
have two different alleles.
• Reference Allele is the base that’s exist in the reference Genome
while the Alternative Allele refers to any base other than the
reference.

4. NGS Variant Calling Overview
What is Variant Calling?
• Identifying single nucleotide polymorphisms (SNPs) and small
insertions and deletion (indels) from next generation sequencing
data.
• Plays an important role in scientific discovery.
Reference
Sample

5. NGS Variant Calling Overview

6. NGS Variant Calling Overview

7. NGS Variant Calling Overview

8. NGS Variant Calling Overview

9. Let’s Look Closer

10. It’s All About The Parameters

11. Variant Calling Experimental Design

12. Why We use the term Clinical Bioinformatics

13. Variant Calling Experimental Design(Clinical Aspects)
• What you have to consider beside the regular parameter setup:
1. Biological nature of the sample and what we are searching for.
2. Patient history retrieval.
3. NGS run QC report (Vertical and horizontal converge, base calling quality,
etc.,…. )
4. Retrieving sample QC records from sample receiving until sequencing.

14. Biological Nature of The Sample (Germline Mutation)
• Germline mutations are present in the
egg or sperm cells that make us.
These mutations are often inherited
from our parents, but can also occur
for the first time in us. Germline
mutations are present in every cell in
our body, and we can pass them on to
our children.

15. Biological Nature of The Sample (Somatic Mutation)
• Somatic mutations, in contrast,
develop in body cells over the
course of life, but do not
involve the egg or sperm cells.
These mutations may cause us
to develop health problems
such as cancer, but we will not
pass the mutation on to our
children, since such mutations
don't occur in the cells involved
in fertilization.

16. Origin of Somatic mutations

17. Patient History Retrieval
• Patient with a mosaic status below your
cut-off.

18. NGS Run QC report (Vertical and Horizontal
Converge)

19. Retrieving Sample QC Records from Sample Receiving
until Sequencing.

20. Variant prioritization

21. Variant prioritization

22. Variant prioritization

23. Variant prioritization

24. Variants effect predictors and annotation resources

25. Reporting

26. Reporting

27. Reporting

28. Reporting

29. Reporting

30. What We Thought
Patient Physician
Sample
Order
Sequence
Tier 1:
Base Calling
Alignment
Variant Calling
Tier 2:
Genome Annotation
Medical Knowledgebase
Tier 3: Clinical Report
EHR

31. Sample
Accessioned
(Tues – Mon)
W1
Cut Scrolls
Tissue
Yes
Sample Received
DNA
Isolation
(Mon – W2)
No
Quality Control:
Nanodrop (all samples)
Qubit (all samples)
1% Gel (all except FFPE)
PCR Ladder (FFPE only)
Gel Chip (FFPE only)
Pass QC?
More Sample
Available?
Aliquot 3ug
in 130ul
Yes
No
Yes
Failed Sample
Report
No
Fragmentation
(Wed – W2)
Quality
Control:
Experion
Chip
Pass
QC?
No
End
Repair
A-tail
Ligate
Adaptors
Quality
Control:
Bioanalyzer
Qubit
Yes
Pass QC?
Library
Amplification
1st
No
Speedvac
Capture
(Fri – W2)
Capture
Wash
(Mon – W3)
Post-
Capture
Amplification
Quality
Control:
Qubit
Yes
Pass QC?
1st
No
2nd
No
3rd
No
4th
No
Dilute Yes
Cluster
Generation
Load HiSeq
(Fri – W3)
1st
Base
Report
Clusters
Generating?
Rehyb
1st
No
Need
Pooled?
Add
10nM
Aliquot
qPCR
(Wed – W3)
Yes
No Quality
Control:
qPCR
Pass QC?
No
Denature
(Thurs – W3)
Yes
Sequence
Read 1
Yes
Index Read
(Read 2)
Turnaround
(Tues – W4)
Sequence
Read 3
(Sat – W4)
Pass
QC?
Quality
Control:
Primary
Analysis
No
Yes
Pass
Analysis?
No
Final Report
Yes
2nd
No
2nd
No
More
DNA?Yes
No
Retrieve
Library
Product
Alignment
(Novoalign)
(Mon – W5)
Sequencing
Output
Data
Variant
Calling
(GATK &
Pindel)
Calculate
Coverage
Variant
Analysis and
Generate
Report
Add Clinical
Interpretation
Draft Report
(Wed – W5)
Review/Edit
by
Pathologist

32. Take Home Massage
• NGS moving to routine clinical diagnosis rapidly but
bioinformatic barriers are the most challenging.
• Clinical validation of NGS assays in cancer is complex and
labor intensive but basic principles remain.
• Your variant calling pipelines and cut-off values should consider
the biological nature of the sample.
• Retrieving all possible history and sample qc report is a must.
• Presence of a well curated clinical annotation database is a
necessity.
• Don’t forget to list down all your steps in the report.

33. References
1. Oliveretal.BioinformaticsforClinicalNextGenerationSequencing.ClinicalChemistry(2015)61:124-135.
2. Gullapallietal.NextGenerationSequencinginClinicalMedicine:ChallengesandLessonsforPathologyandBiomedicalInformatics. J Pathol
Inform (2012) 3:40.
3. Changetal.ClinicalApplicationofAmplicon-BasedNext-GenerationSequencinginCancer.CancerGenetics(2013)206:413-419.
4. Thorvaldsdottiretal.IntegrativeGenomicsViewer(IGV):High-PerformanceGenomicsDataVisualizationandExploration.Brie ngsin
bioinformatics (2012) 14: 178-192.
5. Ulahannanetal.TechnicalandImplementationIssuesinUsingNext-GenerationSequencingofCancersinClinicalPractice.British Journal of Cancer
(2013) 109: 827-835.
6. Perkeletal.SequenceAnalysis101.TheScientist(2011)March1.
7. Targeted Second-Tier Confirmatory Sequencing NBS Pipeline, Nicole Ruiz-Schultz. Utah Department of Health Newborn Screening
Program.
8. Variant Calling with GATK ,Mohammed Khalfan NGS Bioinformatics Workshop NYU. anuary 2018
9. The Clinical Applications of Next Generation Sequencing, Dr. Jonathan Frampton
10. Introduction to Next Generation Sequencing (NGS) ,Andrew Parrish Exeter, 2nd November 2017

34. Thank you

35. Questions