The document discusses the selection and characterization of reference materials for next generation sequencing. It summarizes discussions from a workgroup meeting on choosing reference samples. The workgroup recommended characterizing multiple sequencing platforms and using the sample NA12878, but noted issues with its consent for commercial use. If re-consenting is not possible, the workgroup suggested replacing NA12878 with another sample such as a trio. The workgroup emphasized selecting samples based on characteristics like representing diverse ethnicities and having multiple fully characterized samples, rather than focusing on specific sample IDs.

1. Next
Genera*on
Sequencing:
Reference
Material
(RM)
Selec*on
and
Design
Workgroup
Genome
in
a
BoAle
Consor*um
/
NIST
August
16-­‐17,
2012

2. RM
Scope
• Human
Genome
&
Tumor
Sequencing
• Variant
Types
– SNP
– InDel
/
Subs*tu*on
– CNV
– Structural
variant
| 2

3. Reference
Material
Needed
For
• PlaVorm
valida*on
– Sequencing
System
– Bioinforma*cs/Analysis
Pipeline
• Test
valida*on
– Whole
genome
– Targeted
– Germline
vs.
tumor
• Reagent
valida*on
| 3

4. New
York
State
proposed
dra[
guidelines
for
NGS
clinical
cancer
tests
• …
A
posi%ve/sensi%vity
control
should
be
included
to
verify
analy*c
sensi*vity.
We
suggest
including
an
individually
bar
coded
low
posi*ve
control
near
the
sensi*vity
of
the
assay,
containing
mul*ple
known
soma*c
altera*ons
of
each
kind
to
be
detected,
to
verify
that
low
percentage
gene*c
variants
can
be
detected
in
every
run.
…
• Performance
characteris*cs
for
each
type
of
gene*c
altera*on
the
assay
is
intended
to
detect,
e.g.
SNVs,
indels,
CNVs,
must
be
established
and
validated
• Sequence
a
well-­‐characterized
reference
sample
(e.g.
HapMap
DNA
GM12878)
to
determine
error
rate
and
establish
depth/uniformity
of
coverage
across
all
amplicons.
| 4

5. Associa*on
for
Molecular
Pathology
Comments
to
FDA
UHT-­‐Sequencing
Mee*ng,
June
2011
• …
Performance
of
and
coverage
needs
for
a
given
plaVorm
are
likely
to
differ
depending
on
the
nucleic
acid
and
DNA
regions
analyzed,
the
variants
interrogated,
the
rela*ve
allele
propor*ons
of
par*cular
variants,
…
Evalua*on
should
consider
the
effects
of
rela*ve
GC
content,
homopolymeric
and
other
regions
of
repe**ve
sequence,
homologous
gene
regions
and
DNA
structural
variants,
…
This
necessitates
flexibility
and
individualiza*on
in
the
development
of
valida*on
protocols,
guidelines,
and
controls
on
a
(clinical)
applica*on-­‐by-­‐applica*on
basis.
…
• Assay
controls
should
include
a
range
of
variants,
…
Process
controls
like
NA12876
…
and
the
synthe*c
ERCC
RNA
transcripts
from
NIST
are
examples
of
poten%al
standard
reference
materials.
…
| 5

6. Reference
Material
PorVolio
Whole
Human
Genome
• What
sources
of
RMs
to
consider
– Available
for
research
and
for
profit
– Primary/
cell
line
• What
extent
of
prior
characteriza*on
• Which
ethnici*es,
genders
• Which
muta*ons
need
to
be
present
– Is
medical
relevance
necessary
• Ini*ally
to
have
– ONE
characterized
genome
RM
-­‐
or
– Mul*ple
genomes,
lower
level
of
characteriza*on
• Source
of
commercial
development
and
distribu*on
– Manufactured
under
quality
system
for
diagnos*c
applica*ons
| 6

7. Workgroup
AAendees
• Approximately
25
aAendees
– Federal,
incl.
FDA,
CDA,
NIST
– Lab
accredita*on
– Clinical
reference
labs
– PlaVorm
technologies
– Reference
material
/
reagent
providers
– Research
| 7

8. Workgroup
Summary
(1)
Reference
Material
Use
• Characterize
PlaVorms
&
Methods
– DNA
sequencing
– Exis*ng
&
upcoming
NGS
technologies
– Research
applica*ons
– PlaVorm
&
methods
characteriza*on
for
clinical
diagnos*cs
applica*ons
• Not
intended
as
reference
material
for
– Valida*on
of
specific
muta*ons
in
a
panels
Confidential – Do not copy or distribute | 8

9. Workgroup
Summary
(2)
Selected
Sample
• NA12878
– 6,000
controlled
aliquots
of
10ug
each
on
order
by
NIST
from
Coriell
| 9

10. Workgroup
Summary
(2)
Selected
Sample
• NA12878
– 6,000
controlled
aliquots
of
10ug
each
on
order
by
NIST
from
Coriell
HOWEVER …. | 10

11. Workgroup
Summary
(3)
Consent
• Consent
available
for
– Research
ü
• Consent
not
available
for
– Commercial
• Incl.
altera*ons,
re-­‐distribu*on
– Re-­‐iden*fica*on
through
sequence
data
NA12878 and family requires re-consenting for
commercial use and potential re-identification
through sequence data
| 11

12. Workgroup
Summary
(4)
Sample
&
Consent
• If
re-­‐consen*ng
of
NA12878
not
possible,
replace
with
other
sample
(Trio)
• If
necessary,
do
sample
change
now,
since
early
in
process
of
selec*ng
reference
sample
• New
characteriza*on
of
reference
material
will
be
in
much
greater
depth
and
with
technologies
that
are
more
advanced
than
exis*ng
data
for
NA12878
| 12

13. Workgroup
Summary
(5)
Sample
Characteris*cs
• Sample
characteris*cs
are
more
important
than
selec*on
of
specific
sample
IDs
• More
reference
samples
preferred
over
fewer
samples
– Prefer
8
fully
characterized
samples
at
high
depth
and
corresponding
trios
at
lower
depth
over
4
fully
characterized
samples
plus
trios
| 13

14. Workgroup
Summary
(5)
Sample
Characteris*cs
(cont.)
• High
Priority
– Mul*ple
ethnici*es
• Diversity
in
structural
varia*on
to
stress
systems
– Balanced
female
to
male
ra*o
– Cell
line,
low
passages
• Replenish
supply
• Nice
to
have
– Interracial
marriage
samples
• Controlled
admixture
• Less
cri*cal
– Phenotypic
characteriza*on
• Reference
material
not
for
discovery
– Access
to
RNA
or
*ssues
• No
limitless
supply
of
material
with
iden*cal
characteris*cs
| 14

15. Workgroup
Summary
(6)
DNA
RM
Requirements
• DNA
from
low
passage
cell
lines
– New
batches
from
low
passage
aliquots
• Modify
DNA
purifica*on
in
future
to
keep
step
with
new
NGS
technologies
– Current
purified
DNA
fragment
sizes
are
80-­‐100kb
• OK
for
exis*ng
technologies
– New
nanopore
technologies
may
need
Mbp
fragments
• Agarose
embedding
is
proven
extrac*on
technology
• Consider
footprint
analysis
of
all
batches
prior
to
distribu*on
– Iden*fy
gene*c
dri[,
mix
ups,
….
,
develop
benchmarks
• Reference
material
that
mimics
tumor
sample
characteris*cs
– FFPE
embedded
cells?
| 15

16. Workgroup
Summary
(7)
Sample
Source
Sugges*ons
Most
support
• Personal
Genome
Project
Samples
– Includes
trios
– Use
sequence
data
to
derive
admixture
– hAp://www.personalgenomes.org
– Consent
includes
research
&
commercial
use
and
re-­‐iden*fica*on
Some
support
(if
consent
sufficient)
• HS1011
– Charcot
Marie
Tooth
cell
line
• Lupski
et
al,
NEJM
2010
• MCF10A
– Normal
breast
cancer
• Used
by
Horizon
Dx
to
produce
isogenic
cell
lines
with
cancer
relevant
muta*ons
Other
• African
American
sample
with
70%
sanger
sequence
(ID?)
– No
cell
line
available
– Subject
s*ll
alive
=>
re-­‐consent
&
generate
cell
line?
| 16

17. Reference
Material
PorVolio
Synthe*c
DNA
• What
types
are
most
useful
– Variant
types,
sequence
context,
phasing
• Assess
strengths
&
weaknesses
of
plaVorm
• Test
valida*on
for
exis*ng
and
medically
relevant
variants
• What
size
• How
many
synthe*c
RMs
– PlaVorm
valida*on
– Test
valida*on
• Which
exis*ng
sources
should
be
considered
– Available
for
research
and
for
profit
• Source
of
commercial
development
and
distribu*on
– Manufactured
under
quality
system
for
diagnos*c
applica*ons
| 17

18. Reference
Material
PorVolio
n ce
Synthe*c
DNA
• What
types
are
most
useful
– Variant
types,
sequence
context,
phasing
n
-co fere e
by tele artic ipat
• Assess
strengths
&
weaknesses
of
plaVorm
What
size
vie
w to p
• Test
valida*on
for
exis*ng
and
medically
relevant
variants
•
Resynthe*c
Rnvited iMs
are
• How
many
ll
Aalida*on
– PlaVorm
valida*on
– Test
v
• Which
exis*ng
sources
should
be
considered
– Available
for
research
and
for
profit
• Source
of
commercial
development
and
distribu*on
– Manufactured
under
quality
system
for
diagnos*c
applica*ons
| 18

19. Confidential – Do not copy or distribute | 19

20. Divider slide