METHODS
Patient samples
DNA samples for whole exome sequencing (WES) were obtained from normal
tissue (oral mucosa) and tumor bone marrow. The study was approved by the ethical
research and animal care committee from the Institute of Health Carlos III (CEI PI
32_2009). DNA was extracted by the phenol–chloroform method and the quality and
quantity of purified DNA was assessed by fluorometry (Qubit, Invitrogen) and gel
electrophoresis.
WES and bioinformatic analysis
The preparation of shotgun libraries from the leukemic and non-leukemic
genomic DNA followed the “SureSelect Human All Exon Kit” protocol (Agilent
Technologies, Santa Clara, CA), covering 50 Mb of coding exons (approximately 1.60%
of the genome). One to three µg of high molecular weight genomic DNA from each
sample was fragmented by acoustic shearing on a Covaris S2 instrument. Fractions of
150–300 bp were ligated to Illumina adapters and PCR-amplified for 6 cycles. For exon
enrichment, 300 ng of library were hybridized to SureSelect Human All Exon Capture
kit for 24 h at 65ºC. Biotinylated hybrids were captured, and the enriched libraries
were completed by amplifying with 12 cycles of PCR. The resulting purified DNA library
was applied to an Illumina flow cell for cluster generation and sequenced using the
Illumina Genome Analyzer IIx generating 2x76 base pair, paired-end reads by following
the manufacturer protocols.
Exome variant analysis and biological impact predictions were performed by
RUbioSeq software [1] using default parameters for somatic variation analysis. In
detail, sequencing data were first checked by FastQC for quality control checks on raw
sequence data and then aligned to the human reference genome (GRCh37) using
Burrows-Wheeler alignment (BWA) [2]. That reads unmapped by BWA were realigned
using BFAST [3]. Somatic variants were identified using the Unified Genotyper v2
available at the GATK [4]. For variant calling we used GATK Unified Genotyper v2
applying the ‘‘Discovery’’ genotyping mode and default parameters for filtering. Thus,
SNPs available at dbSNP 132 (hg19) and those reported by the 1000 Genomes Project,
as well as changes present in the matched normal DNA and silent changes, were
filtered out from VCF output files [5]. The GATK QUAL field was employed for ranking
selected somatic variants. Biological impact predictions for detected variants were
obtained from Ensembl Variant Effect Predictor [6]. In order to estimate the damaging
character of the missense mutations, we used two different algorithms: SIFT and
PolyPhen-2. A workflow of WES and the bioinformatic analysis is provide in
Supplementary Figure 2.
We used polymerase-chain-reaction (PCR) amplification and direct DNA
sequencing to validate candidate variants. Because the sensitivity of Sanger, sequence
variants that were reported in less than 20% of the reads could not be included in this
validation phase. PCR products were cleaned using ExoSAP-IT (USB) and sequencing
using ABI Prism BigDye terminator v3.1 (Applied Biosystems) with 5pmol of each
primer. Sequencing reactions were run on an ABI-3730 automated sequencer (Applied
Biosystems). All sequences were manually examined.
IKZF3, ASXL1 and TP53 screening
PCR and Sanger sequencing of IKZF3, ASXL1 and TP53 were performed in 26
additional CML samples (13 CP and 13 BC/NCgR). Primers spanning all coding exons of
IKZF3, exon 13 of ASXL1 and all coding exons of TP53 are reported in Supplementary
Table 3. PCR products were cleaned using ExoSAP-IT (USB) and sequencing using ABI
Prism BigDye terminator v3.1 (Applied Biosystems) with 5pmol of each primer.
Sequencing reactions were run on an ABI-3730 automated sequencer (Applied
Biosystems). All sequences were manually examined.
Supplementary Table 1: SNSs filtering steps of CML WES
Filter
CP
CCyR
BC
Total
3123
7678
3306
Somatic
719
1839
869
Exonic
372
878
412
Consequence*
77
81
66
Quality Control
13
7
15
*Frameshift, stop gain and non-synonymous-deleterious
CML-Phase Position
CP
5_33549323_G/T
CP
5_5232589_G/A
CP
20_31022550_G/T
CP
1_207305072_C/G
CP
4_1388623_-/CA
CP
2_71801335_-/AGGCGG
CP
17_37922621_C/T
CP
4_23825925_23825926delinsCT
CP
1_25824890_A/G
CP
17_7577551_C/T
CP
15_41870343_C/T
CP
21_46197270_T/A
CP
2_145157495_A/C
CCyR
4_1388623_-/CA
CCyR
12_53207608_-/CAG
CCyR
22_29885623_-/AGGAAG
CCyR
5_43613188_TGCCCAAATCCAA/CCyR
13_25671311_TATGA/CCyR
13_21729832_-/TGGAATTT
CCyR
17_7577551_C/T
BC
1_12785494_G/BC
5_33549323_G/T
BC
5_5232589_G/A
BC
X_2835999_CCACGCCGG/BC
20_31022550_G/T
BC
1_207305072_C/G
BC
4_1388623_-/CA
BC
3_75714825_G/BC
17_37922621_C/T
BC
4_23825925_23825926delinsCT
BC
1_25824890_A/G
BC
17_7577551_C/T
BC
15_41870343_C/T
BC
21_46197270_T/A
BC
2_145157495_A/C
Gene Name
ADAMTS12
ADAMTS16
ASXL1
C4BPA
CRIPAK
DYSF
IKZF3
PPARGC1A
TMEM57
TP53
TYRO3
UBE2G2
ZEB2
CRIPAK
KRT4
NEFH
NNT
PABPC3
SKA3
TP53
AADACL3
ADAMTS12
ADAMTS16
ARSD
ASXL1
C4BPA
CRIPAK
FRG2C
IKZF3
PPARGC1A
TMEM57
TP53
TYRO3
UBE2G2
ZEB2
QUAL
Coverage Freq_Var(%) Consequence
503,94
24
8
NON_SYNONYMOUS_CODING
1206,77
96
50
NON_SYNONYMOUS_CODING
323,4
27
48
STOP_GAINED
1060,02
57
61
STOP_GAINED
488,27
122
9
FRAMESHIFT_CODING
320,44
24
20
FRAMESHIFT_CODING
707,81
50
48
NON_SYNONYMOUS_CODING
999,13
113
35
NON_SYNONYMOUS_CODING
2076,54
155
50
NON_SYNONYMOUS_CODING
768,45
47
62
NON_SYNONYMOUS_CODING
244,25
23
4
NON_SYNONYMOUS_CODING
1222,13
87
52
NON_SYNONYMOUS_CODING
954,8
67
55
NON_SYNONYMOUS_CODING
427,91
141
9
FRAMESHIFT_CODING
1437,89
198
5
IN-FRAME
894,64
248
4
IN-FRAME
264,02
91
18
FRAMESHIFT_CODING
225,27
197
9
FRAMESHIFT_CODING
1464,5
81
9
SPLICE_SITE,FRAMESHIFT_CODING
250,69
45
27
NON_SYNONYMOUS_CODING
575,54
187
5
FRAMESHIFT_CODING
633,95
46
50
NON_SYNONYMOUS_CODING
723,43
103
30
NON_SYNONYMOUS_CODING
278,35
29
17
IN-FRAME
390,7
35
49
STOP_GAINED
511,16
53
36
STOP_GAINED
501,39
95
14
FRAMESHIFT_CODING
294,25
243
20
FRAMESHIFT_CODING
618,13
57
39
NON_SYNONYMOUS_CODING
1318,91
117
41
NON_SYNONYMOUS_CODING
1452,52
199
29
NON_SYNONYMOUS_CODING
228,95
17
50
NON_SYNONYMOUS_CODING
142,13
38
20
NON_SYNONYMOUS_CODING
1439,23
104
50
NON_SYNONYMOUS_CODING
808,39
69
45
NON_SYNONYMOUS_CODING
DNA Level (cDNA)
NM_030955.2:c.4291C>A
NM_139056.2:c.1810G>A
NM_015338.5:c.2035G>T
NM_000715.3:c.1071C>G
NM_175918.3:c.324_325insCA
NM_001130987.1:c.3236delinsAGGCGG
NM_012481.3:c.952G>A
NM_013261.3:c.854_855delinsAG
NM_018202.4:c.1928A>G
NM_000546.4:c.730G>A
NM_006293.3:c.2542C>T
NM_003343.5:c.188A>T
NM_014795.3:c.1259T>G
NM_175918.3:c.324_325insCA
NM_002272.2:c.457_458insCAG
NM_021076.3:c.1994_1995insAGGAAG
NM_012343.3:c.330_342del
NM_030979.2:c.976_980del
NM_145061.5:c.1238_1238+1insTGGAATTT
NM_000546.4:c.730G>A
NM_001103170.1:c.584del
NM_030955.2:c.4291C>A
NM_139056.2:c.1810G>A
NM_001669.2:c.701_709del
NM_015338.5:c.2035G>T
NM_000715.3:c.1071C>G
NM_175918.3:c.324_325insCA
NM_001124759.1:c.483del
NM_012481.3:c.952G>A
NM_013261.3:c.854_855delinsAG
NM_018202.4:c.1928A>G
NM_000546.4:c.730G>A
NM_006293.3:c.2542C>T
NM_003343.5:c.188A>T
NM_014795.3:c.1259T>G
Protein Level
p.Pro1431Thr
p.Gly604Arg
p.Gly679*
p.Tyr357*
p.Thr109Glnfs*82
p.Glu1081Glyfs*59
p.Glu318Lys
p.Ser285Lys
p.Lys643Arg
p.Gly244Ser
p.Arg848Trp
p.Asp63Val
p.Leu420Arg
p.Thr109Glnfs*82
p.Gly152_Gly153insAla
p.Lys665_Ala666insGlyArg
p.Ala111Glyfs*23
p.Met326Glyfs*21
p.*413Cysfs*4
p.Gly244Ser
p.Cys195Phefs*17
p.Pro1431Thr
p.Gly604Arg
p.Ala234_Val236del
p.Gly679*
p.Tyr357*
p.Thr109Glnfs*82
p.Arg161Serfs*5
p.Glu318Lys
p.Ser285Lys
p.Lys643Arg
p.Gly244Ser
p.Arg848Trp
p.Asp63Val
p.Leu420Arg
PolyPhen
probably_damaging
probably_damaging
NA
NA
NA
NA
probably_damaging
probably_damaging
probably_damaging
probably_damaging
probably_damaging
probably_damaging
probably_damaging
NA
NA
NA
NA
NA
NA
probably_damaging
NA
possibly_damagin
possibly_damagin
NA
NA
NA
NA
NA
possibly_damagin
possibly_damagin
possibly_damagin
possibly_damagin
possibly_damagin
possibly_damagin
possibly_damagin
SIFT
tolerated
deleterious
NA
NA
NA
NA
deleterious
deleterious
tolerated
deleterious
deleterious
deleterious
deleterious
NA
NA
NA
NA
NA
NA
deleterious
NA
tolerated
deleterious
NA
NA
NA
NA
NA
deleterious
deleterious
tolerated
deleterious
deleterious
deleterious
deleterious
Supplementary Table 2: Somatic SNSs and indels in CML progression
Supplementary Table 3: Primers for ASXL1, IKZF3 and TP53 screening
Primer name
ASXL1-13F
ASXL1-13R
ASXL1-13aF
ASXL1-13aR
ASXL1-13bF
ASXL1-13bR
ASXL1-13cF
ASXL1-13cR
ASXL1-13dF
ASXL1-13dR
ASXL1-13eF
ASXL1-13eR
ASXL1-13fF
ASXL1-13fR
ASXL1-13gF
ASXL1-13gR
IKZF3-2F
IKZF3-2R
IKZF3-3F
IKZF3-3R
IKZF3-4F
IKZF3-4R
IKZF3-5F
IKZF3-5R
IKZF3-6F
IKZF3-6R
IKZF3-7F
IKZF3-7R
IKZF3-8F
IKZF3-8R
TP53-2-3F
TP53-2-3R
TP53-4F
TP53-4R
TP53-5F
TP53-5R
TP53-6F
TP53-6R
TP53-7F
TP53-7R
TP53-8F
TP53-8R
TP53-9F
TP53-9R
TP53-10F
TP53-10R
TP53-11F
TP53-11R
Sequence (5'-3')
GGACCCTCGCAGACATTAAA
AGCTCTGGACATGGCAGTTC
GGTCAGATCACCCAGTCAGTT
CACCACCATCACCACTGCT
CAACTACTGCCGCCTTATCC
GTCGGTGAGGATTCAGGTGT
TGAAGGATCCTGTAAATGTGACC
ATTGCTGTCACTGCCTCCTC
TCACTCTGGACTGTGCCATC
GCAGCAACTGCATCACAAGT
CTCAGTGGAGGCCACTAACC
CATAGCACGGACTTCCTTCTG
CAGAAGGAAGTCCGTGCTATG
GGGACTATGCCCAGTAGCTTT
GGGTCCTCTTAAGGCAAATG
GTTTCCCATGGCCATAATTT
GCTGTTAAGTTTTCCGAAATGG
AAGCACATTCCTCTCTCTTTGG
TGTGTGAAGCTGAAAGTTAAATGG
CTACAATTGCAAGTTTTCCTTGG
TCGTTGTTCATTTCTTGCATTT
TTCTCACGTGGCTGCATTAG
GAGCTTTTCCCCTAGGCATC
ACACTGGGCTCTGAGGAATG
AGTTGTCACAGAGCCCCAAT
ATCTGTCTGCCCCCAGTAAA
GCATCCAACTTCGGACACTT
TTTAACAGAGGTTAAGCTAGGAAAGG
CTTGGCAGTGTTCCCTTTGT
GGCGAGGTCATTGGTTTTTA
CCATTCTTTTCCTGCTCCAC
TCAAATCATCCATTGCTTGG
CCTGGTCCTCTGACTGCTCT
GACAGGAAGCCAAAGGGTGA
GTTTCTTTGCTGCCGTCTTC
GAGCAATCAGTGAGGAATCAGA
CTGCTCAGATAGCGATGGTG
TTGCACATCTCATGGGGTTA
GCACTGGCCTCATCTTGG
GGGATGTGATGAGAGGTGGA
TCTGGCTTTGGGACCTCTTA
GGAAAGAGGCAAGGAAAGGT
AAGCAAGCAGGACAAGAAGC
TGTCTTTGAGGCATCACTGC
AACTTGAACCATCTTTTAACTCAGG
GAAGGCAGGATGAGAATGGA
AAAGCATTGGTCAGGGAAAA
GCAAGCAAGGGTTCAAAGAC
Supplementary Figure 1: Karyotype during CML progression (A) chronic phase: 45, XY,
t(9;22)(q34;q11.2), rob(13;14)(q10;q10) [20]; (B) complete cytogenetic response: 45,
XY, rob(13;14)(q10;q10) [20]; and (C) blast crisis: 44, XY, t(9;22)(q34;q11.2),
rob(13;14)(q10;q10), -17 [5]; 45, XY, t(9;22)(q34;q11.2), +10, rob(13;14)(q10;q10), -17
[5]; 46, XY, t(9;22)(q34;q11.2), +8, +10, rob(13;14)(q10;q10), -17 [3]; 47, XY,
t(9;22)(q34;q11.2), +8, +10,
rob(13;14)(q10;q10), -17, +19 [2]; 45, XY,
rob(13;14)(q10;q10) [5].
Supplementary Figure 2: Sequencing workflow and bioinformatics pipeline for
identification of somatic mutations. After alignment of the sequencing reads from
DNA samples of CML and normal cells to the human reference genome, a series of
filters were applied to discard reads that were not usable for the downstream purpose
of somatic mutation discovery. Sequence variants fulfilling the additional criteria were
subjected to Sanger sequencing validation.
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