mkt0004-2020-02 super chip duplex sequencing …...mkt0004-2020-02 super chip duplex sequencing...
TRANSCRIPT
DuplexSeq™ Tag
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α B
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TTT
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CC
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CT
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Source DNA molecule(Truth)
Top and bottom strands are amplifiedand sequenced. PCR copies are grouped
by unique tag and strand.
Compare top and bottom strands
Duplex consensus eliminates errors
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TwinStrand Duplex Sequencing™ Technology
Sequencing Errors Obscure Truth
Next-GenerationSequencing (NGS)
Single StrandError-Corrected NGS
Duplex Sequencing
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Abstract
Super CHIP: Duplex Sequencing™ Reveals Clonal Dynamics in Bloodthat are Magnitudes more Complex than Previously RecognizedJacob Higgins, PhD1 – Gabriel Pratt, PhD1 – Charles C. Valentine, MS1 – Lindsey N Williams, PhD1 – Masumi Ueda, MD2 – Rainer Storb, MD2 Jerald P. Radich, MD2,3 – and Jesse J. Salk, MD, PhD1,3
TwinStrand Biosciences Inc., Seattle, WA1 – Clinical Research Division, Fred Hutchinson, Seattle, WA2 – Division of Medical Oncology, University of Washington, Seattle, WA3
• Duplex Sequencing detects multiple ultra-low frequency clonal CHIP mutations in all individuals, including the healthy 18-year old control.
• Duplex Sequencing detects drastically more CHIP mutations per individual than in previous studies, and at much lower frequencies.
• Background mutation frequency in the control is 4.7x10-7.• Tracking of ultra-low frequency clonal dynamics between donor and
recipient samples reveals that there is no strong bias for clonal expansion of CHIP variants after HCT.
• Duplex Sequencing identifies enough rare frequency variants to calculate base substitution spectra for all samples. However, there is little difference between donor and recipient spectra. The predominant base substitution signature in all samples is driven by aging.
• The sensitivity of Duplex Sequencing may help determine whether specific CHIP mutations are associated with stronger risk of HM or cardiovascular disease.
Conclusions
Hematopoietic cell transplantation (HCT) is used in the treatment of hematologic malignancies (HM). Clonal hematopoiesis of indeterminate potential (CHIP) is the age-associated process whereby healthy individuals accumulate low-frequency clones driven by mutations in genes recurrently mutated in HM. CHIP is associated with increased risk of cardiovascular disease and transformation into HM.
It has been hypothesized that CHIP clones present in HCT donors might disproportionately proliferate in the recipient due to bottlenecking and a relative growth advantage during immune reconstitution. Here we use ultra-sensitive Duplex Sequencing (DS) of genes associated with HM to investigate whether the clonal makeup of CHIP in prior donors themselves, differs from that of the engrafted donor-derived hematopoietic system multiple years after allogeneic transplant into a recipient.
We identify dozens of mutation-bearing clones in all donor samples sequenced, which are enriched for genes previously associated with CHIP. The extremely high sensitivity and specificity of DS reveals a far more complex landscape of competing ultra-low frequency driver mutations within individuals than observed in any prior studies.
When comparing clonal dynamics between donors and recipients we observe no significant change in clonal makeup, frequency or mutation spectrum, regardless of age. This suggests that HCT does not accelerate CHIP evolution in transplant recipients, which is reassuring from the perspective of stem cell health, secondary cancer risk, and safety of using older donors.
Contact: https://twinstrandbio.com/contact/MKT0004 v1.0
A DuplexSeq™ Adapter has:1.
2.
Identical (or relatable) degenerate tags in each strand.
An asymmetry allowing independent strand identification.
For Research Use Only. Not for use in diagnostic procedures. ©2020 TwinStrand Biosciences, Inc. All rights reserved. All trademarks are the property of TwinStrand Biosciences, Inc. or their respective owners.
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Cou
nt
0.00
0.25
0.50
0.75
1.00
Prop
ortio
n
Substitution Type
C>AC>GC>TT>AT>CT>G
Con
trol
M9
M10
M10
DM
1M
9D M5
M8D M
2M
6D M7
M6
M3
M5D M
4M
4DM
2DM
3DM
7D M8
M1D
Unsupervised heirarchical clustering of the DNA base substitution spectrum in all donors, recipients, and the control. There was no strong base substitution signature unique to HCT recipients, but all donor and recipient samples show an aging signature relative to the 18-year old control. Donors and recipients ranged in age from 49-91 years old at the time of biopsy.
No Unique Base Substitution Signature in HCT Recipients
Total number of multi-count (clonal) variants from donors in known CHIP genes. Variants are colored by predicted effect on the canonical transcript. There was little correlation between gene size and the number of variants detected, arguing against sise-mediated bias. Compared to other approaches used in previous studies, Duplex Sequencing observed many more clonal variants in all patients and genes.
DN
MT3
ATE
T2B
RIN
P3AS
XL1
TP53
RAD
21EZ
H2
CB
LPT
PN11
GAT
A2R
UN
X1W
T1K
RAS
SMC
1AK
ITH
NR
NPK
FLT3
PHF6
STAG
2K
MT2
AID
H2
NR
ASC
EBPA
SMC
3
0
25
50
75
100
125
150
Det
ecte
d cl
onal
exon
ic v
aria
nts
5,000
2,500
0
Targ
eted
exo
nsi
ze (b
p)
Dozens of multi-count (clonal) variants from known CHIP genes were identified in each donor, and only 6 in the control. Variants were colored by predicted effect on the canonical transcript. The majority were missense or nonsense, indicating in vivo selection for those that alter function of the resulting protein.
Donor-Recipient Characteristics
MDS
PNH
CML
AML
AML
CML
AA
AA
AA
CML
6.6
45.7
36.3
38.6
8
32.1
39
37.7
36.9
26.4
PBSC
BM
BM
BM
PBSC
BM
BM
BM
BM
BM
Matched
Matched
Matched
Matched
Matched
Matched
Matched
Matched
Matched
Mismatched
48
23
28
16
42
30
12
22
14
35
84
18
27
14
71
43
13
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12
62
36
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Years fromHCT Disease Stem cell
source HLA-matchRecipient
age at HCT(years)
Donor geat HCT(years)
Age difference betweendonor & recipient
(years)UniquePair ID
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Measure of change in allele frequency in variants shared between donors and recipients. As in previous figures, variants are colored by predicted effect on the canonical transcript. Many variants increased or decreased in frequency, with the majority present at VAF < 1x10-3. Some are unique to donor or recipient (along axes), but there was not a consistent pattern of increased frequency in the recipient. This suggests that HTC does not bias age-associated expansion of clonal variants.
010,00020,00030,000
Mea
nDe
pth
Con
trol
M1
M1D M
2M
2D M3
M3D M
4M
4D M5
M5D M
6M
6D M7
M7D M
8M
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M10
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Clo
nal R
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Varia
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Rare
Var
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6 × 10-6
4 × 10-6
2 × 10-6
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Participant ID
Donor Variant Allele Frequency
Reci
pien
t Var
iant
Alle
le F
requ
ency
1 × 100
1 × 10-1
1 × 10-2
1 × 10-3
1 × 10-4
1 × 10-5
1 × 10-6
1 × 1001 × 10-11 × 10-21 × 10-31 × 10-41 × 10-51 × 10-6
Duplex Sequencing: High Depth, Low Background
Indel - inframeIndel - frameshiftStop LostStart LostStart Gained5'-UTR
3'-UTRSplicingSynonymousNonsenseMissense
Con
trol
M1D
M2D
M3D
M4D
M5D
M6D
M7D
M8D
M9D
M10
D
Participant ID
01020304050607080
Det
ecte
d C
lona
lEx
onic
Var
iant
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CHIP Variants Identified in Every Sample
Abundant Mutations in Canonical CHIP Genes
Complex Donor/Recipient Clonal Dynamics
Barplots of mean Duplex Sequencing depth, frequency of rare variants, and the percent of rare variants that are clonal (multi-count) for all samples in project, including a healthy 18 year old control. Samples were sequenced to at least 24,000x mean Duplex depth. Paired donor and recipient samples generally had a similar proportion of clonal rare variants. Background mutation frequencies were below one-in-one million (1x10-6) for many individuals, including the control. Up to 29% of rare variants were clonal in donors and recipients, with a much lower proportion in the much younger control.
Barplots of mean Duplex Sequencing depth, frequency of rare variants, and the percent of rare variants that are clonal (multi-count) for all samples in project, including a healthy 18 year old control. Samples were sequenced to at least 24,000x mean Duplex depth. Paired donor and recipient samples generally had a similar proportion of clonal rare variants. Background mutation frequencies were below one-in-one million (1x10-6) for many individuals, including the control. Up to 29% of rare variants were clonal in donors and recipients, with a much lower proportion in the much younger control.