SUPPLEMENTARY INFORMATION
1. Materials and Methods
2. Supplementary Figures
3. Supplementary Tables
4. Authors’ Contribution
5. Supplementary References
1. Materials and Methods
Patients. A total of 31 PV patients were collected for the discovery study and the results
obtained were validated in additional two cohorts totaling 59 PV patients. Clinical data
were available for 69 patients across all three cohorts. The collection of blood samples
was performed at the University of Utah, School of Medicine, and was approved by the
Institutional Review Board. Written consent was obtained from all patients in accordance
with the Declaration of Helsinki. The LeukemiaNet criteria for clinicohematologic
response were used to assess treatment responses in patients1.
The allelic fraction of JAK2V617F in GNC and T-cell was determined by WXS for patients
in the discovery study and by deep Ion AmpliSeq sequencing for patients in the
validation study. The WXS data was validated by deep Ion AmpliSeq sequencing. The
1
fraction of 9p aUPD in all patients was analyzed by high-resolution SNP arrays. The
fraction of 9p aUPD in GNC was quantitatively measured as described in our earlier
study3.
Isolation of GNC and T-cells and DNA extraction. Granulocyte (GNC) and
mononuclear cell fractions were isolated according to previously published protocol4. Tcells were positively selected from mononuclear cells by CD3+ MicroBead Kit (Miltenyi
Biotec Inc, Auburn, CA). Genomic DNA was isolated from granulocytes and T-cells
using the Gentra-Puregene Kit (Qiagen, Valencia, CA).
Illumina library construction. Illumina libraries were constructed according to the
manufacturer’s protocol with modifications as described in HGSC website
(https://hgsc.bcm.edu/sites/default/files/documents/Illumina_Barcoded_PairedEnd_Capture_Library_Preparation.pdf). Libraries were prepared using Beckman robotic
workstations (Biomek NXp and FXp models. Briefly, 1 ug of genomic DNA in 100ul
volume was sheared into fragments of approximately 300-400 base pairs in a Covaris
plate with E210 system (Covaris, Inc. Woburn, MA) followed by end-repair, A-tailing
and ligation of the Illumina multiplexing PE adaptors. Pre-capture Ligation MediatedPCR (LM-PCR) was performed for 7 cycles of amplification using the 2X SOLiD
Library High Fidelity Amplification Mix (a custom product manufactured by Invitrogen).
Universal primer IMUX-P1.0 and a pre-capture barcoded primer IBC were used in the
PCR amplification. In total, a set of 12 such barcoded primers were used on these
samples. Purification was performed with Agencourt AMPure XP beads after enzymatic
2
reactions. Following the final XP beads purification, quantification and size distribution
of the pre-capture LM-PCR product was determined using the LabChip GX
electrophoresis system (PerkinElmer).
Exome capture. Four pre-capture libraries were pooled together (approximately 250
ng/sample, 1 ug per pool) and hybridized in solution to the HGSC VCRome 2.1 design1
(42Mb, NimbleGen) according to the manufacturer’s protocol NimbleGen SeqCap EZ
Exome Library SR User’s Guide (Version 2.2) with minor revisions. Human COT1 DNA
and full-length Illumina adaptor-specific blocking oligonucleotides were added into the
hybridization to block repetitive genomic sequences and the adaptor sequences. Postcapture LM-PCR amplification was performed using the 2X SOLiD Library High
Fidelity Amplification Mix with 14 cycles of amplification. After the final AMPure XP
bead purification, quantity and size of the capture library was analyzed using the Agilent
Bioanalyzer 2100 DNA Chip 7500. The efficiency of the capture was evaluated by
performing a qPCR-based quality check on the four standard NimbleGen internal
controls. Successful enrichment of the capture libraries was estimated to range from a 6
to 9 of ΔCt value over the non-enriched samples. Aliquots of enriched libraries (10 nM)
were submitted for sequencing.
Illumina sequencing. Library templates were prepared for sequencing using Illumina’s
cBot cluster generation system with TruSeq PE Cluster Generation Kits (Part no. PE-4013001). Briefly, these libraries were denatured with sodium hydroxide and diluted to 3-6
pM in hybridization buffer in order to achieve a load density of ~800K clusters/mm2.
3
Each library pool was loaded in a single lane of a HiSeq flow cell, and each lane was
spiked with 1% phiX control library for run quality control. The sample libraries then
underwent bridge amplification to form clonal clusters, followed by hybridization with
the sequencing primer. Sequencing runs were performed in paired-end mode using the
Illumina HiSeq 2000 platform. Using the TruSeq SBS Kits (Part no. FC-401-3001),
sequencing-by-synthesis reactions were extended for 101 cycles from each end, with an
additional 7 cycles for the index read. Real Time Analysis (RTA) software was used to
process the image analysis and base calling. Sequencing runs generated approximately
300-400 million successful reads on each lane of a flow cell, yielding 9-10 Gb per
sample. With these sequencing yields, samples achieved an average of 96% of the
targeted exome bases covered to a depth of 20X or greater.
Exome sequencing data processing and quality control. Exome sequence data
processing and analysis were performed using the standard pipelines established at
Human Genome Sequencing Center (HGSC) of Baylor College of Medicine5, 6. Read
sequences were mapped to the human reference genome (GRCh37) by BWA7. All BAM
files were processed to identify duplicates using the Picard and then recalibrated and
realigned by GATK8. Quality control modules were used to compare genotypes derived
from Affymetrix arrays and sequencing data to ensure concordance. Genotypes from SNP
arrays were also used to monitor for low levels of cross-contamination between samples
from different individuals. After PCR duplication removal, we obtained an average of
125x (GNC) and132x (T-cells) coverage of the targeted protein-coding regions. 94.9%
(GNC) and 95.0% (T-cells) of the targeted bases were covered by at least 20 folds.
4
Mutation calling and annotation. The Atlas-SNP2 algorithm was used to identify
somatic single-nucleotide variants in targeted exons9. We also applied Pindel to call
small-to-medium size of insertions and deletions10. A minimum of 4 high-quality
supporting reads and a minimum mutant allele fraction of 0.05 was required for mutation
calling. Somatic mutations and germline variants were annotated using information from
publicly available databases, including dbSNP build 13511, ANNOVAR12 and COSMIC
v5713.
Mutation validation. Mutation validation was done using the Ion Torrent Personal
Genome Machine (PGM, Life Technologies Corporation). Only somatic non-silent
mutations were selected for validation. To make the Ion libraries, amplicons from the
GNC and T-cells were barcoded, pooled, sheared by enzymatic digestion, adaptor ligated,
size selected and amplified according to manufacture’s instructions. The Ion Torrent
sequencing data was analyzed using Torrent Suite Software v3.0. DNA. The average read
depth obtained per base was 2226x and 2034x for GNC and T-cells pools, respectively. A
minimum of 50 high-quality supporting reads and a minimum mutant allele fraction of
0.05 was required to define a validated mutation.
Designing of Ion AmpliSeq arrays. JAK2 for 51 out of 59 PV patients in validation
study was sequenced on the Ion Torrent PGM platform. 45 patients were done using the
Ion AmpliSeq Cancer Hotspot Panel v2 kit (Life Technologies), 17 patients were done
using the Ion Ampliseq custom array and 11 patients were done by both arrays. The
cancer hotspots array includes 2,800 cancer ‘hot-spot’ codons from 50 frequently mutated
5
cancer genes. The amplicons size varied from 80 to 140 base pairs. The Ion Ampliseq
custom array was designed for 42 most frequently mutated genes in myeloproliferative
disorders. The coding exons of 42 selected genes were extracted using UCSC table
browser (hg19) and submitted to Ion AmpliSeq Designer using pipeline version 1.2 using
settings for standard DNA and an amplicon range 125-225 base pairs. The resulting
custom design consists a total of 1202 amplicons. The average amplicon size was 200
base pairs.
Ion Torrent library construction. Ion Ampliseq library kit 2.0 (Cat#4480441, Life
Technologies) consisting of Ampliseq PCR and library preparation reagents was used to
prepare template DNA for sequencing. Ampliseq reactions were performed separately for
Pool 1 and pool 2 for each sample. Each Ampliseq reaction was set up using 10 ng of
DNA as input. Thermo cycling conditions included, initial denaturation for 2 min. at
99°C followed by 16 annealing and extension cycles of 15 s at 99°C and 4 min. at 60°C.
Libraries were prepared using Beckman robotic workstations. Following the Ampliseq
reaction, 2ul of FuPa reagent was added to remove PCR adaptor regions and repair
fragment ends. Ion Xpress™ Barcode Adapters were then ligated to each pool. The Postligation products were purified using Agencourt AMPure XP beads. Thermocycling
conditions were initial denaturation for 2 min. at 98°C followed by 7 annealing and
extension cycles of 15 s at 98°C and 1 Min. at 60°C. Agencourt XP® beads were used to
purify DNA after each reaction step. PCR products were purified using the above SPRI
beads followed by quantification and size distribution using the LabChip GX
electrophoresis system (PerkinElmer). Four to eight samples (8-16 libraries) were
6
sequenced per run on Ion Torrent PGM instrument.
The library templates were prepared for sequencing using the Life Technologies Ion
OneTouch v2 DL protocols and reagents. Briefly, library fragments were clonally
amplified onto Ion Sphere Particles (ISPs) through emulsion PCR and then enriched for
template-positive ISPs. More specifically, PGM emulsion PCR reactions utilized the Ion
OneTouch 200 Template Kit v2 DL (Life Technologies, Part no. 4480285), and as
specified in the accompanying protocol, emulsions and amplification were generated
using the Ion OneTouch System (Life Technologies, Part no. 4467889). Following
recovery, enrichment was completed by selectively binding the ISPs containing amplified
library fragments to streptavidin coated magnetic beads, removing empty ISPs through
washing steps, and denaturing the library strands to allow for collection of the templatepositive ISPs. For all reactions, these steps were accomplished using the Life
Technologies ES module of the Ion OneTouch System, and template-positive ISPs were
quantified using the Guava EasyCyte 5 (Millipore Technologies), obtaining >90%
enrichment efficiency for all reactions. Approximately 20 million template-positive ISPs
per run were deposited onto the Ion 318C chips (Life Technologies, Part no. 4469497) by
a series of centrifugation steps that incorporated alternating the chip directionality.
Sequencing was performed with the Ion PGM 200 Sequencing Kit (Life Technologies,
4474004) using the 440 flow (“200bp”) run format.
Ion PGM sequencing data processing and mutation calling. The PGM sequencing
data was processed using Ion Torrent Suite Software v3.0. Reads were aligned to the
7
genome using TMAP against human reference genome build 37 (NCBI) with default
parameters. Mutations were called using BAM files from the tumor and matched normal
samples. Atlas-SNP51 was run for SNP calling. The variants were further filtered to
remove those supported by less than 5 sequencing reads or presented in less than 8% of
aligned reads. For indels, the variant allele must be supported by at least 10 sequencing
reads. In addition, it is requested that at least one variant had to be Q30 or better and had
to lie in the central portion of the read. Besides, reads harboring the variant must have
been observed in both forward and reverse orientations.
DNA copy number analysis. DNA copy number analysis was done for all 90 patients.
Among them, 50 patients were analyzed by Affymetrix SNP 6.0 array and 40 patients
were analyzed by Illumina610 SNP array. The fraction of the cell population harboring
the 9p aUPD event was quantified by SNP genotyping signal intensities as described in
our earlier study3.
8
2. Supplementary Figures
Supplementary Figure 1
Correlation of the mutational pattern with the prevalence of transformation.
I
II
III
Subgroups
The correlation analysis was done for pooled patients from all 3 studies whose clinical
data are available (n=69). AML, Acute myeloid leukemia; MF, myelofibrosis
transformation. P value for the Chi-square tests is shown.
9
Supplementary Figure 2
1
B Allele Fraction
a
0.8
0.6
0.4
0.2
20
00
0
40 00
00
0
60 00
00
0
80 00
00
10 00
00 0
0
12 00
00 0
0
14 00
00 0
0
16 00
00 0
0
18 00
00 0
0
20 00
00 0
0
22 00
00 0
0
24 00
00 0
0
26 00
00 0
0
28 00
00 0
0
30 00
00 0
0
32 00
00 0
0
34 00
00 0
0
36 00
00 0
0
38 00
00 0
00
00
0
b
V617F
JAK2
Analysis of the relationship of 9p aUPD and JAK2V617F in a PV patient. (a), the aUPD
event observed at chromosome 9p and the detailed view of JAK2 region (lower panel,
colored in red). The distortion of SNP allelic fraction showed complete aUPD across
JAK2 gene. b, IGV view of the JAK2V617F, the mutated base T was colored in red. The
allelic fraction of V617F is 0.24. Our data indicate that the majority of PV clone was
composed of 9p aUPD and only a small subclone also carried JAK2V617F mutation.
10
Supplementary Figure 3
The JAK2 haplotype analysis across 3 subgroups.
The 46/1 risk haplotype10-12 analysis was performed using SNP array data from the
discovery study to determine if there is any association between this risk haplotype and
the progression of GNC from subgroup I to subgroup II. No significant difference in the
frequency of the risk haplotype between the subgroup-I and the subgroup-II patient was
observed (P > 0.05).
11
Supplementary Figure 4
The distribution of 9p aUPD in a representative PV case.
The B allele fraction (B Allele Freq) derived from SNP array is plotted across
chromosome 9. The position on x-axis was sorted ascendingly from p arm to q arm. Mb,
megabase; Probes covering the JAK2 locus were colored in red.
12
Supplementary Figure 5
The candidate genes identified within the aUPD locus on chromosome 9p.
Only those variants that lost the wild-type alleles were counted and those genes recurrent
in at least 3 PV patients were displayed. The upper panel indicates the total number of
patients exhibited loss of heterozygosity of each gene. The bottom panel indicates the
total number of somatic and germline events detected in each gene.
13
3. Supplementary Tables
Supplementary Table 1. Functional annotation of genes identified within the aUPD
locus on chromosome 9p.
Gene
#
Patient
#
nonsilent
FREM1
11
12
FRAS1-related
extracellular
matrix protein
DOCK8
11
9
dedicator of
cytokinesis
KANK1
10
12
Kank proteins
KDM4C
9
11
JmjC domaincontaining
histone
demethylation
protein
CCDC171
9
8
CNTLN
9
7
uncharacterize
d protein
centlein,
centrosomal
protein
ADAMTSL1
8
7
Protein
annotation
a disintegrin
and
metalloprotein
ase with
thrombospondi
n
motif
Protein Function
plays a role in
epidermal
differentiation and is
required for
epidermal adhesion
during embryonic
development
Guanine nucleotide
exchange
factors interact with
Rho GTPases and are
components of
intracellular
signaling networks.
functions in
cytoskeleton
formation by
regulating actin
polymerization; a
candidate tumor
suppressor for renal
cell carcinoma.
specifically
demethylates 'Lys-9'
and 'Lys-36' residues
of histone H3,
thereby
playing a central role
in histone code.
Appears to
associated with the
mother centriole
during G1 phase and
with daughter
centrioles
towards G1/S phase
may have important
functions in the
extracellular matrix
14
Cell
divis
ion
Epigen
etic
regula
tion
Tumo
r
suppr
ession
Transcr
iption
regulati
on
Y
Y
Y
Y
Y
Y
HAUS6
8
5
a subunit of the
augmin
complex
PTPRD
8
2
protein
tyrosine
phosphatase
FOXD4
7
16
Forkheadrelated
transcription
factor
FOCAD
7
8
focadhesin
C9orf66
6
7
KIF24
6
7
uncharacterize
d protein
Kinesins
KCNV2
6
1
Voltage-gated
potassium
channel
subunit
SMARCA2
6
1
a member of
the SWI/SNF
family of
proteins
FAM205A
5
11
C9orf72
5
5
uncharacterize
d protein
uncharacterize
d protein
plays a role in
microtubule
attachment to the
kinetochore and
central spindle
formation.
signaling molecules
that regulate a
variety of cellular
processes including
cell growth,
differentiation,
mitotic cycle, and
oncogenic
transformation
play critical roles in
the regulation of
multiple processes
including
metabolism, cell
proliferation and
gene expression
during ontogenesis.
Potential tumor
suppressor in
gliomas
-
Y
microtubuledependent ATPases
that function as
molecular motors.
They play important
roles
in intracellular
vesicle transport and
cell division
Potassium channel
subunit. Modulates
channel activity by
shifting the threshold
and the half-maximal
activation to more
negative values
is part of the large
ATP-dependent
chromatin
remodeling complex
SNF/SWI, which is
required for
transcriptional
activation of
genes normally
repressed by
chromatin.
-
Y
-
15
Y
Y
Y
Y
Y
DMRT2
5
5
doublesex and
mab-3 related
transcription
factor
Pumilio
domaincontaining
protein
AT-rich
interactive
domaincontaining
protein
KIAA0020
5
5
ARID3C
5
3
INSL4
5
1
insulin-like 4
protein
TEK
5
1
TEK tyrosine
kinase,
endothelial
GLDC
5
0
glycine
dehydrogenase
MOB3B
5
0
kinase
activator 3b
SLC1A1
5
0
FAM154A
4
6
Sodiumdependent
glutamate/aspa
rtate
transporter
uncharacterize
d protein
one of the candidates
for sex-determining
gene(s) on chr 9
Y
-
have roles in
embryonic
patterning, cell
lineage
gene regulation, cell
cycle control,
transcriptional
regulation and
possibly in
chromatin structure
modification.
May play an
important role in
trophoblast
development and in
the regulation of
bone formation
regulates
angiogenesis,
endothelial cell
survival,
proliferation,
migration, adhesion
and cell spreading,
reorganization of the
actin cytoskeleton,
but also maintenance
of vascular
quiescence.
binds to glycine and
enables the
methylamine group
from glycine to be
transferred to the T
protein.
shares similarity
with the yeast Mob1
protein, which binds
Mps1p, a protein
kinase essential for
spindle pole body
duplication and
mitotic checkpoint
regulation.
play an essential role
in transporting
glutamate across
plasma membranes.
-
16
Y
Y
Y
Y
DDX58
4
4
DEAD box
proteins
GLIS3
4
4
Zinc finger
protein
IFT74
4
4
PTPLAD2
4
4
UBAP2
4
4
IL33
4
3
Coiled-coil
domaincontaining
protein
Proteintyrosine
phosphataselike A domaincontaining
protein
ubiquitin
associated
protein
interleukin 33
TTC39B
4
3
TAF1L
4
2
DENND4C
4
1
TOPORS
4
1
tetratricopepti
de repeat
protein
transcription
initiation factor
TFIID subunit
DENN/MADD
domain
containing 4C
Tumor
suppressor
p53-binding
protein
putative RNA
helicases, may play
important roles in
granulocyte
production and
differentiation,
bacterial
phagocytosis and in
the regulation of cell
migration
functions as both a
repressor and
activator of
transcription
-
Y
Responsible for the
dehydration step in
very long-chain fatty
acids (VLCFAs)
synthesis
Cytokine that binds
to and signals
through IL1RL1/ST2,
Induces T-helper
type 2-associated
cytokines
May act as a
functional substitute
for TAF1/TAFII250
during male meiosis,
when sex
chromosomes are
transcriptionally
silenced
functions as an
ubiquitin-protein E3
ligase, Probable
tumor suppressor
involved in cell
growth, cell
proliferation and
apoptosis that
regulates p53/TP53
stability through
ubiquitin-dependent
degradation. May
regulate chromatin
modification through
17
Y
Y
sumoylation of
several chromatin
modificationassociated proteins.
KIAA2026
4
0
uncharacterize
d protein
interferon
epsilon
methylthioaden
osine
phosphorylase
IFNE
3
4
MTAP
3
3
ANKRD18B
3
2
BNC2
3
2
DMRT3
3
1
NOL6
3
1
AQP3
3
0
water channel
protein
aquaporin 3
DMRTA1
3
0
IFNB1
3
0
doublesex- and
mab-3-related
transcription
factor
Fibroblast
interferon
KIAA1432
3
0
ankyrin repeat
domaincontaining
protein
zinc finger
protein
basonuclin-2
doublesex and
mab-3 related
transcription
factor
Nucleolar RNAassociated
protein
Connexin-43interacting
protein
plays a major role in
polyamine
metabolism and is
important for the
salvage of both
adenine and
methionine.
-
Probable
transcription factor
specific for skin
keratinocytes. May
play a role in the
differentiation of
spermatozoa and
oocytes
May regulate
transcription during
sexual development
associated with
ribosome biogenesis
through an
interaction with prerRNA primary
transcripts.
Involved in skin
hydration, wound
healing, and
tumorigenesis.
May be involved in
sexual development
Has antiviral,
antibacterial and
anticancer activities
Required for
phosphorylation and
localization of GJA1
18
Y
Y
SLC24A2
3
0
a member of
the
calcium/cation
antiporter
superfamily of
transport
proteins
mediate the
extrusion of one
Ca2+ ion and one K+
ion in exchange
for four Na+ ions.
Y, related; -, the function of this protein has not been determined.
19
4. Authors’ contribution
L.W. conducted the major bioinformatics analyses of the sequencing and SNP array data,
wrote and revised the manuscript. D.A.W. and J.T.P. conceived the study, supervised the
implementation of the research plan, reviewed and revised the manuscript. S.I.S. prepared
genomic DNA samples. J.T.P. and K.H. collected, interpreted clinical data and obtained
necessary regulatory documents and Informed Consents from studied subjects. K.W.
contributed the AmpliSeq array design and analysis pipeline. D.M.M. managed the
sequencing and mutation validation pipeline. K.W provided further analyses of highresolution copy-number arrays and assisted with interpretation of data. J.D. contributed
the pipeline of WXS. J.G.R. managed the pipeline for sequencing data mapping,
realignment and recalibration. D.M.M. managed the production pipeline. R.A.G.
reviewed and revised the manuscript.
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