1. Use
cases
of
GIAB
Reference
Materials
NIST,
HSPH,
Claritas,
NCI,
MSSM,
Personalis,
Qiagen
Genome
in
a
Bo>le
Consor?um

2. Preliminary
uses
of
high-­‐confidence
NIST-­‐GIAB
genotypes
for
NA12878
• NIST
has
released
several
versions
of
high-­‐
confidence
genotypes
for
its
pilot
RM
• We’ve
collected
some
examples
of
how
people
are
using
these
genotypes

3. •
•
is pharmacogenomics — the use ample, three recently published a step fu
of genomic information to iden- clinical trials raise questions consider
tify the right drug at the right about the clinical utility of using conditio
dose for each patient. More than pharmacogenetic information in not, in
120 FDA-approved drugs have the initial dosing of vitamin K premium
pharmacogenomics information anatagonists.3
however,
in their labeling, providing imThe FDA based its decision to
So
you’ve
sequenced
my
differences grant marketing authorization for of genom
portant details about
insuranc
genome.
How
well
did
the drug and, in the Illumina instrument platform ance, or
you
in response to
do?
some cases, recommending ge- and reagents on their demonThe l
netic testing before prescribing.2 strated accuracy across numer- use of g
– FDA
approval…
NIST
work
omacogenomics potential of phar- ous human chromosomes. Preci- medicine
n
But the full is largely unreal- 19 genomic segments, spanning of this
developing
the
most
accurate
of the logistic chal- sion and reproducibility across Court ru
ized, because
interpreta?on
of
a
human
suitable instruments, users, days, and re- lecular Pa
lenges in obtaining
ENGLA ND were also demonstrated. that isol
genomic information in a
genome,
coupled
with
The NEWtimely agent lots JOURNAL of MEDICINE
a
enough fashion to guide
The marketing authorization of DNA ca
NIST
Reference
Material
prescribing. Placing genomic information
platform for clinicurrently
being
delectronic medical record a sequencingprobably expand the decision
eveloped,
in the
cal use will
access to
enabled
FDA
to
assess
the
of per- incorporation of genetic informa- but also
would facilitate this kind
performance
of
the
sonalized medicine. If the pa- tion into health care. But even for the in
sequencer
stient’s entire genome were part the most promising technologies quencing
ubmi>ed
for
marke?ng
aof his or her medical record, cannot fully realize their poten- fore the
pproval.
NIST
work
with
FDA
helps
answer
the
ques?on…
Perspective
Paper
Describing
NIST-­‐GIAB
n JOURNAL of
The NEW ENGLA NDengl j med nejm.org MEDICINE
2
Characteriza?on
of
NA12878:
First FDA Authorization for Next-Generation Sequencer
Nature
Biotechnology,
accepted
The New England Journal of Medicine
Francis S. Collins, M.D., Ph.D., and Margaret A. Hamburg, M.D.
Downloaded from
DOI:
arXiv:1307.4661
[q-­‐bio.GN]
nejm.org at FDA Biosciences Library on November 20, 2013. For personal use only. No

4. HSPH
–
Brad
Chapman
Comparing
variant
callers
h>p://bcbio.wordpress.com/2013/10/21/updated-­‐comparison-­‐of-­‐variant-­‐detec?on-­‐
methods-­‐ensemble-­‐freebayes-­‐and-­‐minimal-­‐bam-­‐prepara?on-­‐pipelines/

5. Freebayes
SNP
calls
changed
very
li>le
in
2013
h>p://www.bioplanet.com/gcat/reports/1933-­‐westleouzm/variant-­‐calls/illumina-­‐100bp-­‐pe-­‐exome-­‐150x/bwamem-­‐
freebayes-­‐0-­‐9-­‐10-­‐131226/compare-­‐1934-­‐akckizzzfr-­‐1931-­‐laqgzjytqw-­‐1935-­‐xwckffckoa/snp/group-­‐quality

6. Freebayes
indel
calls
improved
in
2013
h>p://www.bioplanet.com/gcat/reports/1933-­‐westleouzm/variant-­‐calls/illumina-­‐100bp-­‐pe-­‐exome-­‐150x/bwamem-­‐
freebayes-­‐0-­‐9-­‐10-­‐131226/compare-­‐1934-­‐akckizzzfr-­‐1931-­‐laqgzjytqw-­‐1935-­‐xwckffckoa/indel/group-­‐quality

7. Feedback
from
MoCha
lab
in
NCI
• We
built
a
targeted
amplicons
NGS
assay
for
detec?ng
muta?ons
in
clinical
tumor
specimens
• To
assess
the
assay’s
specificity,
we
compared
84
runs
of
CEPH
NA12878
data
from
our
assay
with
NIST’s
consensus
variant
list
(VCF
v2.15)
• We
observed
a
high
overall
concordance
with
a
few
FP
variants
in
homopolymeric
regions
unique
in
our
plahorm
• We
concluded
that
NIST
GIAB
is
a
useful
reference
standard
to
evaluate
assay
specificity

8. Personalis
–
Categorizing
exome
regions
h>p://www.personalis.com/assets/files/posters/ashg2013/Towards_a_medical-­‐grade_exome.pdf

9. Genome
in
a
Bo>le
@
Mount
Sinai
Michael
Linderman
Icahn
Ins?tute
for
Genomics
and
Mul?scale
Biology
Dept.
of
Gene?cs
and
Genomic
Sciences

10. Ongoing
clinical
pipeline
valida?on
Reference
Materials
SNP
Array
Technical
replicates:
NA12878
NA12891
NA12892
NA18507
NA10080
Sanger
Concordance
Analysis
Genome
in
a
Bo>le
Pla?num
Genomes

11. Evalua?ng
and
tuning
variant
calling
&
filtering
Measure
overall
analy?cal
performance
Tune
VQSR
threshold
senng
to
inflec?on
point
We
evaluate
a
set
of
NA12878
technical
replicates
against
GIAB
for
each
new
pipeline
version

12. GIAB
Use
at
Qiagen
(Frederick,
MD)
• Use
GIAB
false
posi?ve
sites
to
quickly
iden?fy
PCR
ar?facts
in
reads
from
PCR-­‐enriched
samples.
• Compare
accuracy
of
PCR-­‐enrichment
amplicon
sequencing
to
accuracy
of
hybridiza?on-­‐capture
whole-­‐exome
sequencing.
• Tune
variant
calling
pipeline
for
good
balance
between
sensi?vity
and
specificity.
• Compare
variant
calling
methods,
of
course!

13. iden?fy
PCR
ar?facts
quickly
• example:
sequenced
fragment
was
primed
by
a
PCR
primer-­‐
dimer
strand
formed
in
earlier
PCR
cycles
5-GGACCTGTGGGTGGGTAAC-3
oligo intended for chr1 locus
|||||||||||xxxx
3-GACACCCACCCGAGGTT-5 oligo intended for chr5 locus
5-GGGTCTGTGGGTGGGCTCCAA-3 DNA sample (chr5 locus)
|| |||||||||||||||||
3-CCTGGACACCCACCCGAGGTT-5 dimer product primes 4 bp upstream
AC
false positive variant called

14. compare
amplicon
enrichment
to
hybridiza?on
capture
enrichment
• characterize
panels
for
mul?plex-­‐PCR
enrichment
• compare
to
exome
capture
read
set
from
Mount
Sinai
Medical
School
(105x
coverage,
36.6Mb)
read
set
indel
TPR
PPV
GeneRead
662
Kb
46.9
0.94
0.92
Mount
Sinai
37
Mb
snp
FPR
x
1E6
7.6
0.89
0.99
GeneRead
662
Kb
9.1
0.60
0.79
Mount
Sinai
37Mb
0.7
0.75
0.98

15. tune
variant
calling
pipelines
• generate
ROC
curves
to
compare
Strelka
and
MuTect
matched
tumor/normal
• “tumor”
here
is
8,
16,
36,
and
100%
spike-­‐in
of
NA12878
DNA

16. NIST GIAB Confident Calls as the Gold Standard
● Community resource to which all can contribute and access
○ Group-specific or internally-generated gold standards have unknown
methods, origin, and curation.
○ NIST GIAB confident calls allows public access and contributions.
● Claritas Genomics uses this callset for:
● Technology feasibility, research and development
○ test new technologies, protocols, reagents, methods, etc.
● Validation and verification
○ assay validation and verification for clinical use
● Critical attributes:
○ a large number of confident true negative positions!
○ confident region allows for scale or genome-scale sensitivity and
specificity analysis.
○ part of a large well-characterized pedigree
○ a trusted source of truth
CONFIDENTIAL
CLARITAS GENOMICS

17. Other
use
cases?
• What
other
types
of
uses
might
people
develop?