Visualizing NGS variants called from VCF files critical for proper clinical/research interpretation.

1. Visualizing Genomic
Variants and Annotations
is Vital for Accurate
Interpretation
April 23, 2015
Gabe Rudy
@gabeinformatics
VP Product Management and Engineering
Golden Helix

2. My Background
 Golden Helix
- Founded in 1998
- Genetic association software
- Analytic services
- Thousands of users worldwide
- Over 800 customer citations in journals
 Products I Build with My Team
- SNP & Variation Suite (SVS)
- SNP, CNV, NGS tertiary analysis
- Import and deal with all flavors of upstream data
- VarSeq
- Annotate and filter variants in gene panels, exomes and
genomes for clinical labs and researchers.
- GenomeBrowse (Free!)
- Visualization of everything with genomic coordinates.
All standardized file formats.

3. Visualization of Variants to Aid Interpretation
 Variants + Genomic Context
- Where it is in gene
- Annotations that match, don’t match
- Other variants in cohort
- Nearby variants in cohort/population
 Alignment Evidence
- BAM files provide more than is in VCF
 Variant Representation
- Multi-Allelic Sites
- Allelic Primitives
- Left-Alignment
- Combination!

4. My Exome Variants

5. My OTC Variant

6. X:38226614 - G/A
• Novel in all Population Catalogs but ExAC’s ~60K exomes

7. X:38226614 - G/A
• Recent Addition to ClinVar:
• 2013-05-09 G/A - Untested with Disease Unspecified
• 2014-03-03 G/A - Pathogenic with not_provided
citing:

8. X:38226614 - G/A
• Cited PubMed article was on ResearchGate, Hiroki Morizono contacted
• Provided full text and lots of interesting backstory on OTC
• “If you are able to eat all the steak you want, you may have the mutation; it would
appear to be a hypomorphic allele (and a very mild one at that)”
• “Is possible that the late onset case that [was] identified may have been someone
who was having a very bad day, and several things went poorly for them.”
• “The R40H mutation, there was a grandfather or granduncle who was affected who
ate whatever he wanted, and seemed unaffected while the proband had several
episodes.”

9. X:38226614 - G/A
• Most likely partial penetrance, with potential risk of triggering with shock event
• The Glycine is conserved down to Opossum (Platypus, Zebafish has a Alanine)

10. The Reference Sequence

11. Splice Mutation

15. Transcripts

16. Reference Sequence Versus Gene Sequence
EMG1 on GRCh37
 “Gap” of the mRNA coding sequence versus reference seq:
 Handled differently by 3 different “gene alignments”

17. Reference Sequence Versus Gene Sequence
EMG1 on GRCh38
 Reference sequence patched, no gap
 Alignments agree

19. Left-Align

21. Left-Align Delta F508 to Make it Match

22. Left-Align Annotations
 Using a Smith-
Waterman
algorithm to left-
align variants
from public
databases show
non-obvious
differences
 NGS alignment
and variant
calling always
left-aligned
 Left-align your
database so they
can be annotated

23. Allelic Primitives

24. My Son’s de Novo

25. Exome Sequencing in Consumer Genomics
 Exomes done as part of Pilot
Program
 80x coverage
 Raw data with no interpretation
Erin
JIA
Gabe
(me)
Ethan

26.  asdf

29. NM_002626.4:c.1877G>C in PFKL
 NP_002617.3:p.Arg626Pro missense mutation
 Predicted damaging by 4/5 functional predictions
 VEST3: 0.948, GERP++: 4.59
 ExAC and 1kG have a G>A, but G>C is novel
 Variants in region are extremely rare (G>C ExAC 4 of 122,364 alleles) – 0.003%
 No ClinVar variants for gene
 OMIM entry has no known disease association
 PubMed search shows few recent articles: Most recent 1998 paper showed
- phosphofructokinase (PFKL) overexpressed in Down syndrome (DS)
- Transgenic PFKL mice had an abnormal glucose metabolism with reduced clearance
rate from blood and enhanced metabolic rate in brain.

30.  d

31.  d
35 LoF Variants, None Homozygous

32. Thank you
 Heidi Rehm – Chief Laboratory Director at
Laboratory for Molecular Medicine,
PCPGM
 Hiroki Morizono – Children’s National
 Reece Hart – Computational Biologist,
Invitae (now 23andMe)
 Greta Linse Peterson – Director of Product
Management and Quality, Golden Helix

33. Questions?