SUPPLEMENTARY DATA
Single-cell genetic analysis reveals the composition of initiating clones and
phylogenetic patterns of branching and parallel evolution in myeloma
Lorenzo Melchor, Annamaria Brioli, Christopher P Wardell, Alexander Murison, Nicola E
Potter, Martin F Kaiser, Rosemary A Fryer, David C Johnson, Dil B Begum, Sanna Hulkki
Wilson, Gowri Vijayaraghavan, Ian Titley, Michele Cavo, Faith E Davies, Brian A Walker,
Gareth J Morgan
TABLE OF CONTENTS
Supplementary Methods
p.2
Supplementary Tables
p.7
Table S1. Median depth and coverage of all samples included in the current analysis.
p.7
Table S2. List of primers and Taqman assays for mutation detection (SNP Genotypingbased) per sample
p.8
Table S3. Number of sorted, analyzable and filtered cells and tumor cells per patient in
the current study.
p.16
Table S4. List of TaqMan® Copy Number Assays
p.17
Table S5. List of the 124 SNVs described in the analysis of a plasma cell leukemia
patient at presentation and relapse and in two engrafted-myelomas (Table available on
additional Excel file)
p.17
Supplementary Figure Legends
p.18
Figure S1. Analysis pipeline of the current study
p.18
Figure S2. Single-cell genetic analysis confirms the mutation frequency shown in exome
sequencing
p.18
Figure S3. Mutational analysis of case 90827 depicts a pattern of linear tumor evolution
p.19
Figure S4. Linear evolutionary pattern shows founder myeloma initiating clone
p.19
Figure S5. Tumor evolutionary pattern with two KRAS mutations acquired in a linear
stepwise process
p.20
Figure S6. Branching evolutionary pattern in case 90482 using only mutation information
p.21
Figure S7. Phylogenetic natural history of presentation-relapse PCL patient
p.21
Supplementary References
p.22
1
SUPPLEMENTARY METHODS
Presentation-relapse patient: case history
A 61 years old gentleman was diagnosed with plasma cell leukemia and spinal cord compression.
Following surgery and radiotherapy of the spine he was treated with two cycles on DT-PACE
(dexamethasone, thalidomide, cisplatin, adriamycin, cyclophosphamide and etoposide) followed by
autologous stem cell transplantation (ASCT) achieving a complete remission. The disease relapsed 1
year after ASCT and was treated with 5 cycles of bortezomib, cyclophosphamide and dexamethasone
(CVD). Despite an initial response the disease evolved and eventually became resistant to treatment.
At the time of progression after CVD bone marrow was collected and plasma cells were detectable in
the peripheral blood. The patient received third line treatment with bortezomib, thalidomide,
cyclophosphamide and dexamethasone (CVTD) for two cycles and later with lenalidomide,
cyclophosphamide and dexamethasone (CRD). Both line of treatment failed to obtain a response and
the patient was referred to palliative care 24 months after diagnosis.
Whole-exome sequencing and pipeline analysis
Whole-exome sequencing (WES) was previously performed in the cases belonging to Myeloma IX
trial. An additional case (11/010) as well as a patient with presentation and relapse sample were
added to this study by performing the same WES approach as before.1 Briefly, libraries were prepared
from tumor and non-tumor DNA from the same patient, and were sequenced to identify acquired
single-nucleotide variants (SNVs) and indels in the tumor sample. We used 50ng of genomic DNA to
capture the exome using the SureSelect Human All Exon 50Mb target enrichment set (Agilent
Technologies, Wokingham, UK). DNA fragmentation, end-repair, A-tailing, ligation to Illumina adaptor
sequences (Illumina, London, UK), PCR amplification, hybridization overnight to the SureSelect
Human All Exon baits (Agilent) and subsequent bioinformatic analyses were carried out as before.1
Purified libraries were sequenced on a GAIIx sequencer (Illumina). Alignment to GRCh37 was
performed using first the BWA2 and then the Stampy3 aligners. Resulting BAM files underwent base
score quality recalibration using the GATK4,5 and duplicate reads were removed using Picard
(http://picard.sourceforge.net/). All BAM files were realigned together using the GATK indel realigner
to minimize artifacts between samples. SNVs were called using MuTect.6 Detected variants were
2
required to meet the following criteria: minimum 10x depth in both tumor and normal samples, at least
one supporting read in both directions, mean base quality of >26 for bases at the SNV site and a
mean mapping quality of >50 for all reads at that site. The site was required to be unique according to
the mapability data available from the UCSC genome browser. Finally, an additional filter was applied
for C>A|G>T SNVs to take into account somatic change artifacts reported by Costello et al7.
SNP Array profiles from a previous study8 and ExomeCNV data from the BAM files, were used to infer
copy number values. These data were combined with the proportion of non-reference reads in the
tumor sample in order to calculate the proportion of cells containing each SNV as previously
described.
Owing to the high identity between the human and mouse genomes, two strict precautions were taken
to avoid false positives in the xeno-transplanted samples. First, aligned xeno-transplant BAMs were
filtered to remove any reads containing more than a single mismatch to the reference genome.
Second, private SNVs in xenograft samples were treated as artifacts and removed. As a result of this
filtering, the median depth for xenograft samples after deduplication was 31x (range 15-47) with an
average of 58.4% of the targeted exome covered at a minimum depth of 20x (Supplementary Table
S1). All SNVs were manually inspected using IGV to verify that they were both correctly called and
correctly annotated as being present or absent in each of the tumor samples.
Clone assessment and filtering approach for translocations, mutations and copy number
aberrations
To define tumor subclones and the most plausible clonal phylogeny, we used minor modifications to
the filtering strategy for wells with low quality DNA amplification and subclones without a minimum
number of cells, used elsewhere.9 Supplementary Figure S1 summarizes the main filtering steps
(characters in pink). First, DNA which had undergone STA process was loaded into the Fluidigm
Dynamic Arrays, but not all rows in the Fluidigm Heatmap gave positive amplification for the
interrogated assays. We removed from further analysis all those cells/rows which failed to amplify in
at least one of the reference SNP assays (for mutation call) or in any of the reference copy number
assays (for copy number calling). Additionally, Ct values >30 cycles were flagged as NA.
3
For mutation and translocation calling (red square in Supplementary Fig. S1), we transformed Ct
results for mutation and translocation assays into binary (1, presence of mutation; 0 wild type)
considering the assay replicates per cell (2-4 replicates). Results from all the interrogated mutations
per cell were combined and cells with NA values were removed from subsequent analyses. The total
number of sorted analyzable tumor single-cells was calculated. The threshold to define subclonal
populations was established in at least 5% of the total of analyzable tumor cells. All cells in fractions
below such thresholds were removed from further analysis.9
For copy number aberrations, cells were initially filtered out if normal reference regions failed to
amplify. CopyCaller software was used to estimate the calculated copy number values (cCNV). Nine
comparisons were performed in Copycaller, comparing the 3 reference control region assays versus
the 3 test region assays. This provided a total of nine cCNVs for each tested region in any of the
analyzed cell per patient. cCNVs were removed if confidence intervals were below 0.5. Additionally,
cCNV>4.5 were not considered, as the number of replicates did not ensure statistical accuracy for
copy number estimation.10 The weighted mean of the cCNVs (weighted copy number values, wCNV)
was calculated only when at least 3/9 replicates were available. If less than 3, such region was
considered NA in that specific cell. wCNV below 1.5 and above 2.5 were considered as genomic loss
(-1) and gain (1) respectively; whereas intermediate values mean no change (0). Then, we plotted
histograms with the wCNV for each cell in all plates. Reference array histogram was characterized for
the accumulation of cells within the normal parameters, but a group of cells provided wCNV >2.5 and
<1.5 as a consequence of the qPCR technical variation.10,11 For each region, we calculated the
percentage of normal cells misleadingly displaying gains or losses and used the highest frequency as
a threshold to set the minimum number of cells required to be considered subclone in copy number
aberration analyses (subclonal thresholds). Qualitative values for each region and cell (loss, no
change, gain) were combined and cells with NA value for any region were removed from further
analysis. The total number of analyzable tumor single-cells for copy number aberrations was then
calculated and subclonal thresholds for each tested chromosomal region were applied. 9 Hierarchical
clustering illustrated the different aberration patterns in clonal populations. Copy number aberrant
subclonal populations were considered when cells were grouped in numbers higher than each of the
subclonal thresholds established per chromosomal region. All cells not fulfilling the criteria were
filtered out.
4
We then combined both filtered mutation and copy number information for each cell, and followed an
additional filtering process with the same criteria for both features.9 A total number of filteredanalyzable tumor cells was estimated and clonal phylogenies and percentages could be accurately
established.
Genetic algorithm to detect clonal lineages in the comparison presentation-relapse patient
samples and xenograft-samples
A genetic algorithm was implemented using the GA package for R 12 to calculate the most
parsimonious assignments of mutations to clonal lineages. Custom population generation, fitness,
mutation, and crossover functions were written.
The population being optimized consisted of a series of vectors, each composed of three parts. The
first n elements (where n is a predicted number of lineages present in the sample) are integers
identifying the parent of the lineage ni, with 0 indicating that this lineage is the root lineage. The next n
x m elements (where m is the number of cases observed) are integers, initialized between 0 and 5,
but with no upper bound following mutation, representing the relative abundance of lineage ni in case
mj. The remaining l elements (where l is the number of mutations observed) are integers from 1 to n
representing the earliest lineage in which each mutation li occurs. The population generation function
produces vectors with exactly one root lineage, where the lineages can be represented as a directed
acyclic graph (DAG), and with all remaining elements assigned randomly.
The fitness function calculates the predicted proportion of the total sample that should contain each
mutation based around the assumption that a mutation should appear in the lineage it first occurs in
and all children of that lineage. The proportion of each lineage in case mj is computed first as the
relative abundance of lineage ni in case mj divided by the sum of all ni in case mj. Then for each
mutation the proportions of all lineages containing that mutation are summed. The absolute value of
the difference between the predicted proportion and the experimentally observed proportion of each
mutation in each case is summed. The fitness function then returns -1 times the sum of these values
as a score to be optimized.
The mutation function assumes that the predicted lineages, abundances or mutation calls can change
with equal probability. In the case that lineages are changed, a completely new DAG for the lineages
5
is produced and replaces the original. In the case that relative abundances are changed, a random
relative abundance is incremented or decremented by 1 (in the case that a decrement would reduce
abundance below 0, the abundance is incremented instead). In the case that a mutation is changed, a
random mutation is assigned a new lineage at random. The crossover function uses single point
crossover. However, to ensure that lineages are always representable as a DAG the crossover
cannot occur during the first n elements of a vector.
The remaining parameters required for the GA function were selected as follows: population size:
100, elitism: 2, mutation rate: 0.8, tournament selection, iterations: 1500, type: binary. The function
was iterated over n=5:12 predicted lineages, and the best scoring individual was selected. R scripts
for this modified algorithm are available on request.
IRF4 HRM analysis
PCR followed by HRM was performed using the Type It HRM Kit (QIAGEN) on a Rotorgene Q realtime cycler (QIAGEN). Primers were designed to span a segment of 140 base pairs: IRF4 forward: 5’AAGAGCAATGACTTTGAGGAACTG-3’; IRF4 reverse: 5’-TGGCTGCCTCTGTTAGGTGA-3’. The
PCR reaction comprised 7 pmol of combined forward and reverse primer mix, 20 ng of DNA, 5 µl of
2x HRM Master Mix and 3.3 µl of RNAse-free water. PCR conditions were a 95°C hold (5 minutes),
followed by 40 cycles of 95°C (10 seconds), 55°C (30 sec) and 72°C (10 sec). HRM analysis was
conducted from 65°C to 95°C at 0.1°C increments. Data were analyzed using the Rotorgene Q series
software 1.7. PCR products with a variant melt profile by HRM were purified using the QIAquick PCR
purification kit (QIAGEN) and sequenced using the BigDye terminator v3.1 chemistry (Life
Technologies) on a 3500 Genetic Analyzer (Life Technologies).
6
SUPPLEMENTARY TABLES
Supplementary Table S1. Median depth and coverage of all samples included in the
current analysis.
Human/
Mouse
Sample
Tumor/
normal
Median
depth
20X
coverage
Duplication
Human
90003
Normal
52
78.9
17.3
Human
90003
Tumor
51
79.4
13
Human
90468
Normal
53
83.4
27.7
Human
90468
Tumor
69
84.2
22.1
Human
90482
Normal
59
83.2
29
Human
90482
Tumor
61
82.1
29.9
Human
90827
Normal
58
86.5
40
Human
90827
Tumor
53
79.2
20
Human
90879
Normal
52
81.1
21.1
Human
90879
Tumor
41
74.5
11.6
Human
11010
Normal
61
86.8
34.3
Human
11010
Tumor
45
80.4
39.2
Human
pclPres
Normal
82
84.4
14.2
Human
pclPres
Tumor
36
72.7
34
Human
10/022*
Tumor
113
93.2
49.4
Mouse
Xeno1
Xeno1
15
34.6
45.4
Mouse
Xeno2
Xeno2
47
82.1
31
Median
depth
(range)
20x
coverage
(range)
Median
Duplication
59
(36-113)
82
(72.7-93.2)
26.9
(11.6-49.4)
31
(15-47)
58.4
(34.6-82.1)
38.2
(31-45.4)
*Sample at relapse of the pclPres patient.
7
Supplementary Table S2. List of primers and Taqman assays for mutation detection (SNP Genotyping-based) per sample
Reference SNP-based assays, used in all tested multiple myeloma samples
Assay name
rs346172
Location
Intronic variant
Chromosome
19
Position
13843233
Sequence
GCCCAAAATGCCCCAAACGCCATCCGGACCATAAAAGTGA[T/C]GGCGATAGTGTTGGACTTAGGAGCTCAAAGATGCTAACCT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Variant Reporter (FAM-NFQ)
CAAACGCCATCCGGACCATA
GCATCTTTGAGCTCCTAAGTCCAA
CACTATCGCCATCACTTT
ACTATCGCCGTCACTTT
Assay name
rs909895
Location
Intronic variant
Chromosome
20
Position
8747242
Sequence
AGAAACAGGAGGGAATCCCACTGCTAGGTTCTTTAGAGCT[A/G]GAGCAAGCAACAGTTCAAAATGTATCGAACGTGCAATAAG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Variant Reporter (FAM-NFQ)
GGAATCCCACTGCTAGGTTCTT
TTCAGTAGAATCTTATTGCACGTTCGA
TTGCTTGCTCTAGCTCTA
CTTGCTCCAGCTCTA
Sample 90003 - Analysis of translocation and nine mutations
Translocation
t(11;14)
Derivative chromosome
Der11(CCND1)::IGHD2-2
Chr14, chr11
GATGCTATTTCTCTTGGGAAAGCACCTAGGAGTGGGTTGTATGGTAAAAGTATATTTAACTTTGTAAGAAACTGCCAAATTACTGCAGAATGGCTGT
ACACCTGCCGCTAAAGCAGCTGGTACTACTACAATATCCTCACAGTGACACGAGCCCCCACAAAATCCTCCTGTCCCCGCGGGAGTCACTGAGTC
Sequence
CCCTCTTGCTGTCTCTGGCTAGTTCTCCTGCTGATACTATGATTTCCAGGGGGTTTTTGTCTGAAACTCAGGGTGTGTGGGAGAGGACTCTGAGC
CCAGTGCTGTACAGGGGGCTCCTCCTTTGTCCTGGGGG
Forward Primer
Reverse Primer
Taqman Probe (FAM-NFQ)
GGCCCCAGTAGTAGCAACCA
CCGGCACTGGCAAGGAT
TAGTCGTCAGTGCATGACAACTCAGATGTTCTC
Gene name
KRAS
Nucleotide change
c. 182A>G
aa change
p. Q61R
Chromosome
12
Position
25380276
Sequence
AGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTCT[T/C]GACCTGCTGTGTCGAGAATATCCAAGAGACAGGTTTCTCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CATGTACTGGTCCCTCATTGCA
GATGGAGAAACCTGTCTCTTGGAT
TGTACTCCTCTTGACCTGC
ACTCCTCTCGACCTGC
Gene name
KRAS
Nucleotide change
c.34G>C
aa change
p. G12R
Chromosome
12
Position
25398285
Sequence
TGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[C/G]AGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GCTGTATCGTCAAGGCACTCTT
AGGCCTGCTGAAAATGACTGAATAT
TTGGAGCTGGTGGCGTA
TTGGAGCTCGTGGCGTA
8
Gene name
ACTG1
Nucleotide change
c. 50G>C
aa change
p. C17S
Chromosome
7
Position
79479331
Sequence
CACGGCTCGGGGAGCGTCGTCCCCAGCAAAACCAGCTTTG[C/G]ACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CGTCGTCCCCAGCAAAAC
CGCGCTGGTCATTGACAAT
CATGTGCAAAGCTG
CATGTCCAAAGCTG
Gene name
CYP4A22
Nucleotide change
c. 356C>T
aa change
p. S119F
Chromosome
1
Position
47607261
Sequence
AAAGCCCTTTCTTACTTTTCAGACCCGAAATCCCATGGAT[C/T]CTACAAATTCCTGGCTCCACGGATTGGTATGTGTGCAAAC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCCTTTCTTACTTTTCAGACCCGAAA
TCCTAGTTTGCACACATACCAATCC
CAGGAATTTGTAGGATCCAT
AGGAATTTGTAGAATCCAT
Gene name
C7ORF23
Nucleotide change
c. 10T>C
aa change
p.F4L
Chromosome
7
Position
86848810
Sequence
CTGTTGTCCAGGCCACTGGTGCCGTAGGTCCTGGTAGCAA[A/G]GTCCTCCATTTTGGGGTTTCTTCACTTTCCCCAAGCCACT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCACTGGTGCCGTAGGT
GGTCATGCTGTCTTGCTTTTAAGTG
AAAATGGAGGACTTTGCTAC
ATGGAGGACCTTGCTAC
Gene name
EGR1
Nucleotide change
c.1169A>G
aa change
p. H390R
Chromosome
5
Position
137803307
Sequence
TTCAGCCGCAGCGACCACCTCACCACCCACATCCGCACCC[A/G]CACAGGCGAAAAGCCCTTCGCCTGCGACATCTGTGGAAGA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCACCTCACCACCCACAT
GCAGGCGAAGGGCTTTTC
CCGCACCCACACAGG
CGCACCCGCACAGG
Gene name
IRF4
Nucleotide change
c. 368A>G
aa change
p. K123R
Chromosome
6
Position
394972
Sequence
CTGGTTGAGCGGAGCCAGCTGGACATCTCAGACCCGTACA[A/G]AGTGTACAGGATTGTTCCTGAGGGAGCCAAAAAAGGTAGG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GGAGCCAGCTGGACATCTC
GGCTCCCTCAGGAACAATCC
AGACCCGTACAAAGTGTA
AGACCCGTACAGAGTGTA
Gene name
LRRIQ1
Nucleotide change
c.2116T>A
aa change
p.S706T
Chromosome
12
Position
85450687
Sequence
AGAAAGCTCCATGGTATCTAAAGAAGTCAACTCTCTTAAA[T/A]CTGAGATTAGAAATATTTCAGAAAAATGCCATGAAAATGC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GCAGAAAGCTCCATGGTATCTAAAGA
TTCAGGTGCATTTTCATGGCATTTT
ATTTCTAATCTCAGATTTAAG
ATTTCTAATCTCAGTTTTAAG
9
Gene name
MUC16
Nucleotide change
c. 42872A>G
aa change
p. H14291R
Chromosome
19
Position
8971520
Sequence
CACATGGATGTCCACCAACTGGTAGGTGGAGCCCAGCCAA[T/C]GGAATGAGGCATTCAGGGTCTTATCTAGAAAGACTTGCT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GATGTCCACCAACTGGTAGGT
CTGGTGGAGCAAGTCTTTCTAGAT
CCAGCCAATGGAATGA
CAGCCAACGGAATGA
Sample 90468 - Analysis of translocation and five mutant genes
Translocation
t(11;14)
Derivative chromosome
Der14 IGHE::chr11(PPP6R3)
Chr14, chr11
CCTTGATAGGTGACCTGGCAGGTGTAGGTGCGGTCTGACAGCCAGTGCTTCTGGCTGAGGGTGAGCTCGCTTTGTGTGGAGGCCAGCTCACCCT
CCTGCGTGGTAGAGGCGGTGGACAAGTCCACGTCCATGACCTGCCCGTCCTCCAGCCAGGTGATGTTGATAGTCCCTGGGGTGTAAGGTAGGAA
Sequence
CTGCTGATTTGAGCCATTGAAGGTTATCACTGCTCAGCTGGACTAGTAAAAGCAAATAAGTACGGTTGCTTTTCCTTGCTAGGACAGACCTGGGAC
CGCATCTCCAGGACAGGGGAGGCTCC
Forward Primer
Reverse Primer
Taqman Probe (FAM-NFQ)
CCAGCCAGGTGATGTTGATAGTC
AGTCCAGCTGAGCAGTGATAACC
TGTAAGGTAGGAACTGCTGATTTGAGCCATTG
Gene name
Sequence
BRAF
c. 1799T>A
aa change
p. V600E
Chromosome
7
Position
140453136
CAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTC[A/T]CTGTAGCTAGACCAAAATCACCTATTTTTACTGTGAGGTC
Forward Primer
TGGGACCCACTCCATCGA
Gene name
Nucleotide change
BDP1
Reverse Primer
CATGAAGACCTCACAGTAAAAATAGGTGAT
Nucleotide change
c.2041G>A
aa change
Wild-type Reporter (VIC-NFQ)
CTAGCTACAGTGAAATC
p. V681I
Chromosome
Mutant Reporter (FAM-NFQ)
TAGCTACAGAGAAATC
5
Position
70797473
Sequence
AATGGAGACTACAGAGAGAGAGAATCCAGAAGCTGAAACT[G/A]TATCTGTGTGAGTATTCAGGAAGTAGTAAAAAAAAAAAAA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
AGTAGAAAACAAATTATTACCATCATT
GGAGACTACAGAGAGAGAGAATCCA
AAGCTGAAACTGTATCTG
AAGCTGAAACTATATCTG
CCTAAGAGTT
Gene name
DIS3
Nucleotide change
c. 2765A>C
aa change
p. K922T
Chromosome
13
Position
70797473
Sequence
GATTTTTGTTGAATATAGCTATTTTCCAAGCTTCATCTTC[T/G]TTTTCTTTGGTCCATTAAGGTCCATGTTTGAAGTATCAGT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCAGTCTTTGAAGATTTTTGTTGA CCTACTGATACTTCAAACATGGACC
AGCTTCATCTTCTTTTTCTT
CTTCATCTTCGTTTTCTT
ATATAGCT
TT
10
Gene name
TP53
Nucleotide change
c. 448C>G
aa change
p.R150G
Chromosome
17
Position
7577094
Sequence
GGCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCC[G/C]GTCTCTCCCAGGACAGGCACAAACACGCACCTCAAAGCTG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CTTTCTTGCGGAGATTCTCTTCCT
GCTTTGAGGTGCGTGTTTGTG
TGCGCCGGTCTCT
TGCGCCCGTCTCT
Gene name
WWC2
Nucleotide change
c. 377C>T
aa change
p. A126V
Chromosome
4
Position
184129241
Assay failed during experiment analysis
Sequence
ACACAGAAGGAACTGTACCATGTGAAGGAGCAGAGGCTGG[C/T]GCTGGCCCTGGATGAATACGTGCGATTAAATGATGCCTAT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GAAGGAACTGTACCATGTGAAGGA GGCATCATTTAATCGCACGTATTCA
CAGCGCCAGCCTC
CCAGCACCAGCCTC
Sample 90482 - Analysis of six mutant genes
Gene name
NRAS
Nucleotide change
c. 182A>G
aa change
p. Q61R
Chromosome
1
Position
115256529
Sequence
TGTCCTCATGTATTGGTCTCTCATGGCACTGTACTCTTCT[T/C]GTCCAGCTGTATCCAGTATGTCCAACAAACAGGTTTCACC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
TGGTCTCTCATGGCACTGTACT
GGTGAAACCTGTTTGTTGGACATAC
ACAGCTGGACAAGAAG
ACAGCTGGACGAGAAG
Gene name
ACAD10
Nucleotide change
c. 828C>A
aa change
p. D276A
Chromosome
12
Position
112150439
Sequence
TGGAAATTCCGAAAGATTCCTTGCAGAAGTACCTCAAAGA[C/A]TTACTGGGTATCCAGACCACAGGTATGTGGGCTTCTTTCA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CGAAAGATTCCTTGCAGAAGTACCT AAACATGAAAGAAGCCCACATACCT
ACCCAGTAAGTCTTTG
ACCCAGTAATTCTTTG
Gene name
IL6R
Nucleotide change
c. 404G>C
aa change
p. G135A
Chromosome
1
Position
154403028
Assay failed during experiment analysis
Sequence
TTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGTGGG[G/C]TCCTCGGAGCACCCCATCCCTGACGACAAAGGCTGTGCTC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCCCTCAGCAATGTTGTTTGTG
CCAAGAGCACAGCCTTTGTC
CTCCGAGGACCCCACT
TCCGAGGAGCCCACT
Gene name
Sequence
PCDH15
Nucleotide change
c.2272_2273delinsTA
aa change
p. G46I
Chromosome
10
Position
56138657
CACATTATCCTTTAAAGAAAGTTCTATGGTGGGGTCTGGT[CC/AT]TCCAGCAGTCCCTTTGATCAGCATGTTGTCCACCAGAATT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
AACACCCAGTAATCCACATTATCCTTT
TGGTGGACAACATGCTGATCAAA
ACTGCTGGAGGACCAG
ACTGCTGGAATACCAG
11
Gene name
STK24
Nucleotide change
c. 1220C>T
aa change
p. S407F
Chromosome
13
Position
99109461
Sequence
CCTCTGGAGCCGCTGCACGAGCTGGGCCACCATGGTGTCG[G/A]AGATGCCAGGGCACGCCTCCTCCGCTAGGTAGATGGCCCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCGCTGCACGAGCTG
GCCATCTACCTAGCGGAGGA
CATGGTGTCGGAGATG
ATGGTGTCGAAGATG
Gene name
TRPA1
Nucleotide change
c. 2272C>T
aa change
p. H758Y
Chromosome
8
Position
72951123
Sequence
TTAAAATTTGAAATTACCGTGGTATCTAGTATTTCTGAAT[G/A]ATCACTAGTTTCATTGATGATGCCAGTTGAGTTGAAAGCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
TCAACTCAACTGGCATCATCAATGA
CTTTATTTTAGAAAGTTAAAATTT
CTAGTATTTCTGAATGATCACT
CTAGTATTTCTGAATAATCACT
GAAATTACCGTGGT
Sample 90827 - Analysis of five mutations
Gene name
KRAS
Nucleotide change
c. 182A>G
aa change
p. Q61R
Chromosome
12
Position
25380276
Sequence
AGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTCT[T/C]GACCTGCTGTGTCGAGAATATCCAAGAGACAGGTTTCTCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CATGTACTGGTCCCTCATTGCA
GATGGAGAAACCTGTCTCTTGGAT
TGTACTCCTCTTGACCTGC
ACTCCTCTCGACCTGC
Gene name
ATM
Nucleotide change
c. 428A>G
aa change
p. N143S
Chromosome
11
Position
108106493
Sequence
GATTCATCTAATGGTGCTATTTACGGAGCTGATTGTAGCA[A/G]CATACTACTCAAAGACATTCTTTCTGTGAGAAAATACTGG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GTGAAAGATTCATCTAATGGTGCT TTTCACACCAGTATTTTCTCACAGA
CTGATTGTAGCAACATACT
TGATTGTAGCAGCATACT
ATTTACG
AAGA
Gene name
GMEB1
Nucleotide change
c. 478A>T
aa change
p. M160L
Chromosome
1
Position
29023446
Sequence
TGAACTGGTACTTCTTGTCTCCCTTTGCACAGGAAAATG[A/T]TGGACTCCGGACAGATTGATTTTTACCAACATGACAAAGT
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
TGGTACTTCTTGTCTCCCTTTGC GGAGCAAACTTTGTCATGTTGGTAA
ACAGGAAAATGATGGACTC
CAGGAAAATGTTGGACTC
12
Gene name
KLK8
Nucleotide change
c.356A>G
aa change
p. H119R
Chromosome
19
Position
51503389
Sequence
CAGGGATGCCTGGTCACGCAGTTGAAGAAGCATCAGATCA[T/C]GGTTGTGGTCCTCCACATCGCTGCTGTTGTAGCAGGGGTG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CGGGTACATGTGCCTGTCAT
CCCCGCTCCCTCCTACA
AAAGGGCGAAGGTC
AAAGGGCAAAGGTC
Gene name
POLE
Nucleotide change
c. 776G>A
aa change
p. R259H
Chromosome
12
Position
133253974
Sequence
ACAAGCAAAACTTACAGGTCGTTCAACAAGGTCATCTCGG[C/T]GGGTGATTTCTACCGGAAAAGCATTTCCTCGGTATCTGAC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
AACTTACAGGTCGTTCAACAAGGT
AATGTCAGATACCGAGGAAATGCTT
ATCACCCGCCGAGATG
AAATCACCCACCGAGATG
Sample 90879 - Analysis of five mutations
Gene name
KRAS
Nucleotide change
c. 183A>C
aa change
p. Q61H
Chromosome
12
Position
25380275
Sequence
CAGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTC[T/G]TGACCTGCTGTGTCGAGAATATCCAAGAGACAGGTTTCTC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCTCATGTACTGGTCCCTCATTG
GATGGAGAAACCTGTCTCTTGGAT
CACTGTACTCCTCTTGACCT
ACTGTACTCCTCGTGACCT
Gene name
KRAS
Nucleotide change
c. 199A>C
aa change
p. M67L
Chromosome
12
Position
25380259
Sequence
CAAAGAAAGCCCTCCCCAGTCCTCATGTACTGGTCCCTCA[T/G]TGCACTGTACTCCTCTTGACCTGCTGTGTCGAGAATATCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCCTCCCCAGTCCTCATGTA
ACACAGCAGGTCAAGAGGAGTA
CTGGTCCCTCATTGCACT
TGGTCCCTCAGTGCACT
Gene name
ATR
Nucleotide change
c. 5965A>C
aa change
p. T1989P
Chromosome
3
Position
142212087
Assay failed during experiment analysis
Sequence
GCTCGACCATGGATTAACATGTTCTTACCCTCAGGTGGGG[T/G]TTCATTTTCAGGAAAACATAATTCAACACCTTTTTGAAGA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GCATAGCTCGACCATGGATTAACAT
CACCAGGCACTAATTGTTCTTCAAAA
CCTGAAAATGAAACCCCAC
CCTGAAAATGAACCCCCAC
13
Gene name
PCDH1
Nucleotide change
c.3325C>T
aa change
p. R1109C
Chromosome
5
Position
141233996
Sequence
CGGGTACATGTGCCTGTCATGGCGACATCAGGCAAAGGGC[G/A]AAGGTCTGTAGGAGGGAGCGGGGAAGGACAATTGTCAGAC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CGGGTACATGTGCCTGTCAT
CCCCGCTCCCTCCTACA
AAAGGGCGAAGGTC
AAAGGGCAAAGGTC
Gene name
PCLO
Nucleotide change
c. 3542C>G
aa change
p. S1181X
Chromosome
7
Position
82595562
Sequence
TTTTTGATCTGTGGTTACCATAGGAGGAATTTTTTCCATT[G/C]ATAGTGTTTCCTTTACTTTTTCCAGAATGACTTTTTCAGC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
TGATCTGTGGTTACCATAGGAGGAA
GGAAGCTGAAAAAGTCATTCTGGAA
AAAGGAAACACTATCAATGG
AAGGAAACACTATGAATGG
Sample 11/010 - Analysis of translocation and seven mutant genes
Translocation
t(11;14)
Derivative chromosome
Der11(CCND1)::IGHD3-22
Chr14, chr11
GGGCGCCTACTCCCCACCACTTGGTCTGAGAGGGGCTGGGGCCAGCCGGAAGGCCAGGGGTCTGTGCGATACGGTAACCACTAC
TATCATAGTAATACCACAGTGACACAGACCTCACTTCAAACCTACCGCCAGGCCTGGGGAAACCCGGGATGTCCAGGGCTGACCTG
AGGAGGCAGCAGGGCCCCGAGGGGAGGCTGTGGGCCCAGCGCTCTCAGGTCTACTGCAGGGACACTCGGGTCTGTCCCTCGCTT
Forward Primer
Reverse Primer
Taqman Probe (FAM-NFQ)
CGCCTACTCCCCACCACTTG
GGTCTGTGTCACTGTGGTATTACTATGA
TGCGATACGGTAACCAC
Sequence
Gene name
KRAS
Nucleotide change
c. 183A>C
aa change
p. Q61H
Chromosome
12
Position
25380275
Sequence
CAGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTC[T/G]TGACCTGCTGTGTCGAGAATATCCAAGAGACAGGTTTCTC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
CCTCATGTACTGGTCCCTCATTG
GATGGAGAAACCTGTCTCTTGGAT
CACTGTACTCCTCTTGACCT
ACTGTACTCCTCGTGACCT
Gene name
NRAS
Nucleotide change
c. 182A>G
aa change
p. Q61R
Chromosome
1
Position
115256529
Sequence
TGTCCTCATGTATTGGTCTCTCATGGCACTGTACTCTTCT[T/C]GTCCAGCTGTATCCAGTATGTCCAACAAACAGGTTTCACC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
TGGTCTCTCATGGCACTGTACT
GGTGAAACCTGTTTGTTGGACATAC
ACAGCTGGACAAGAAG
ACAGCTGGACGAGAAG
14
Gene name
ABCA4
Nucleotide change
c. 3294C>T
aa change
p. R1098C
Chromosome
1
Position
94508353
Sequence
TTACCTGAGCGATACTTCAGGAGCAGATCCCAGATTGAGC[G/A]TCTCGAGTAAGGGTCCACCCCAGAGGTGGGTTCGTCCAGA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GAGCGATACTTCAGGAGCAGATC
CAAGGTGGTGATTCTGGACGAA
CAGATTGAGCGTCTCGAG
CAGATTGAGCATCTCGAG
Gene name
FAT
Nucleotide change
c. 6080T>G
aa change
p. I2026M
Chromosome
4
Position
126336196
Sequence
TCTACAACTTGGTTGTTCAAGTGCATGACCTGCCACAGAT[T/G]CCAGCCTCCAGATTCACAAGCACTGCTCAAGTCTCCATTA
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GGTTGTTCAAGTGCATGACCTG
CTTGAGCAGTGCTTGTGAATCTG
AGGCTGGAATCTGT
AGGCTGGCATCTGT
Gene name
HSP90AA1
Nucleotide change
c. 405G>A
aa change
p.G135S
Chromosome
14
Position
102552221
Assay failed during experiment analysis
Sequence
ACTTTCTCAGCAACCAAATAAGCAGAATAAAAACCAACAC[C/T]GAACTGGCCAATCATAGAGATATCTGCACCAGCCTGCAAA
Forward Primer
TCACAGTTACTTTCTCAGCAACCAA
Gene name
IRF4
Reverse Primer
AGGCTGGTGCAGATATCTCTATGA
Nucleotide change
c. 368A>G
aa change
Wild-type Reporter (VIC-NFQ)
TTGGCCAGTTCGGTGTT
p. K123R
Chromosome
Mutant Reporter (FAM-NFQ)
TTGGCCAGTTCAGTGTT
6
Position
394972
Sequence
CTGGTTGAGCGGAGCCAGCTGGACATCTCAGACCCGTACA[A/G]AGTGTACAGGATTGTTCCTGAGGGAGCCAAAAAAGGTAGG
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GGAGCCAGCTGGACATCTC
GGCTCCCTCAGGAACAATCC
AGACCCGTACAAAGTGTA
AGACCCGTACAGAGTGTA
Gene name
PCSK6
Nucleotide change
c.463G>A
aa change
p.R154Q
Chromosome
15
Position
101938646
Sequence
CACCTTTTTAATGCCATACTCGAAAGCCTGCTTAGCCAGT[C/T]GGCCGGGCCCGTCCACCGTCTTGCCGTCGTCGTCCGGCCC
Forward Primer
Reverse Primer
Wild-type Reporter (VIC-NFQ)
Mutant Reporter (FAM-NFQ)
GCCATACTCGAAAGCCTGCTTAG
GGACGACGACGGCAAGA
CCCGGCCGACTGG
CCGGCCAACTGG
15
Supplementary Table S3. Number of sorted, analyzable and filtered cells and tumor cells
per patient in the current study.
Mutation analysis
Patient
sample
90003
90468
90482
90827
90879
11/010
Sorted
Fixed
Cells
n
75
(100%)
73
(100%)
243
(100%)
241
(100%)
113
(100%)
82
(100%)
Analyzable
Tumor Cells
n (%)
68
(90.6%)
70
(95.9%)
211
(86.8%)
241
(100%)
104
(92.0%)
49
(59.8%)
Filtered
Tumor
Clone
Cells
n (%)
51
(68.0%)
57
(78.1%)
173
(71.2%)
216
(89.6%)
101
(89.4%)
40
(48.78%)
Merged mutation and copy
number aberration
analysis
Filtered
Cells with
Filtered
Assessed
Tumor
assessed
Tumor
tumor cells
Clone
mutations and
Clone
Cells
CNA
Cells
n (%)
n (%)
n (%)
n (%)
Not applicable,
no CNA present in tumor sample
65
48
42
42
(89.0%)
(65.8%)
(57.6%)
(57.6%)
206
204
170
135
(84.8%)
(83.9%)
(70.0%)
(55.6%)
235
202
179
158
(97.6%)
(83.8%)
(74.3%)
(65.6%)
99
79
70
60
(87.6%)
(69.9%)
(61.9%)
(53.1%)
Not applicable,
no CNA present in tumor sample
Copy number aberration
analysis
% refers to the percentage of assessed tumor cells from the initial number of sorted cells,
once filtering criteria has been fulfilled.
CNA, copy number aberration.
16
Supplementary Table S4. List of TaqMan® Copy Number Assays.
Target
Region
Target
Gene
1q21.3e
CKS1B
4q31.1d
MAML3
5p15.2c
CTNND2
6q26a
MAP3K4
8p21.2b
EBF2
8q22.1c
CCNE2
Assay Names
Hs01945685_cn
Hs06604158_cn
Hs02711416_cn
Hs02995147_cn
Hs02707346_cn
Hs01378348_cn
Hs05984957_cn
Hs06098736_cn
Hs06039168_cn
Hs02034242_cn
Hs02678111_cn
Hs01526097_cn
Hs05059431_cn
Hs06223840_cn
Hs06202676_cn
Hs02924866_cn
Hs00704915_cn
Hs05064083_cn
Target
Region
Target
Gene
10q24.1b
LCOR
11q22.2a
BIRC2
13q14.2b
RB1
16q12.1c
CYLD
17p13.1c
TP53
21q22.11b
GCFC1
Assay Names
Hs00384966_cn
Hs00280627_cn
Hs02714907_cn
Hs02270456_cn
Hs02418165_cn
Hs02808252_cn
Hs02920052_cn
Hs03026562_cn
Hs02956242_cn
Hs00284962_cn
Hs02488587_cn
Hs02276538_cn
Hs06424630_cn
Hs06423639_cn
Hs00362931_cn
Hs05536540_cn
Hs05555996_cn
Hs02547302_cn
Supplementary Table S5. List of the 124 SNVs described in the analysis of a plasma cell
leukemia patient at presentation and relapse and in two engrafted-myelomas. Note columns
with the name of the mutated gene, the protein mutation, the cancer-cell fraction frequency
(CCF) on each of the four analysed samples, the sample in which a mutation is found (Venn
Group) and the clone in which a mutation is firstly acquired (Clonal lineage column).
(Provided as an Excel file)
17
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1. Analysis pipeline of the current study. Single-cells were
sorted on the basis of PI+ nuclei staining into DNA lysis buffer in 96 well-plates. This was
followed by specific target amplification using genotyping, translocation detection and copy
number assays and subsequent DNA dilution. Both diluted DNA and Taqman assays were
added to Fluidigm 96.96 Dynamic Arrays, and loaded and mixed with the HX Controller
machine. qPCR reaction was performed in a BioMarkHD equipment to get results for all
tumor and reference arrays. First, we analysed qPCR results for translocation and mutation
calling using filters for low Ct values, NA values, and subclones with less than 5% of tumor
cells. Second, qPCR results were interrogated for copy number calls using Copycaller and
estimating weighted means for the calculated copy number values (wCNV). We used the
normal cells from the reference array to establish thresholds for the minimum number of
cells with a particular genomic aberration to constitute a subclone, and applied such
thresholds to the hierarchical clustering of the cells with available data. Filters were used for
cCNV and wCNV values, low replicates, NA values, and subclones in the tumor arrays not
reaching the established thresholds. Information from both filtered-mutant and copy number
tumor subclones were combined and further filtered out using a similar approach. Finally, the
most plausible tumor phylogeny including both mutations and copy number aberrations is
produced. Hierarchical clusters were firstly performed in Rock platform and further
customized using R scripts.
Supplementary Figure S2. Single-cell genetic analysis confirms the mutation
frequency shown in exome sequencing. (a-f) Column barplots with the mutation
frequencies called by exome-sequencing or FISH (white columns), or by single-cell analysis
considering only filtered tumor cell clones (black columns) for the series of studied samples.
18
Mutation/aberration frequencies calculated by exome-sequencing or FISH respectively, and
by single-cell genetic approaches, had a positive Pearson’s correlation (r) for 5/6 of cases
(p<0.05). Sample 90468 is the case with the lowest correlation value and no statistical
significance, likely due to lack of tested mutations at low frequencies.
Supplementary Figure S3. Mutational analysis of case 90827 depicts a pattern of
linear tumor evolution. (a) Hierarchical clustering of 216 patient cells using only data for
presence/absence of somatic mutations. Grey means absence, red means presence of
mutation or SNP. Note four tumor clones can be noted according to their mutational profile.
(b) Tumor phylogeny using only mutation information reveals a linear sequential process of
acquisition of each mutation (colored stars) from the earliest ancestor clone, which only has
ATM c.428A>G mutation, to the latest clone, arisen as a product of KRAS c.182A>G
mutation. Absolute numbers and percentage of cells composing each subclone are depicted.
(c) Histograms for the weighted means of the calculated copy number values (wCNV) for
8q/21q in all sorted tumor single-cells. Red columns mean cells displaying loss (wCNV≤1.5);
grey, no change (1.5<wCNV<2.5); blue, gains (wCNV≥2.5). Note the higher frequency for
+8q/+21q in the tumor as compared with the reference cell histograms, but still there is a
minor fraction of cells with normal copy number values. The combination of wCNV data and
mutational profiles allows depicting a comprehensive case phylogenetic history (see Figure
2c).
Supplementary Figure S4. Linear evolutionary pattern shows founder myeloma
initiating clone. Genetic study of case 90468 shows: (a) Thumbnail heatmap of the
Fluidigm Array used for this case displaying 57 filtered tumor single-cells. Layout is as in
Figure 2. Zoomed images show the two clonal patterns seen in this case. 4 cells are
depicted in each. Note clone 1 only has positivity for t(11;14) probes (black arrow) and
19
reference SNPs. Clone 2 shows amplification for t(11;14), reference SNPs and the four
additional mutated genes interrogated. (b) Two clones compose the clonal phylogeny of
case 90468. Clone 1 (7% of cells) may represent the founder myeloma initiating clone as
only carries the Ig gene translocation. The steps for clone 1 to give rise to clone 2 cannot be
defined. Mutations could have been sequentially or simultaneously acquired. Additionally,
this clone 2 may simply be the clone with the best clonal selection fitness out of clone 1
potential progeny. Mutations in BRAF, TP53, and DIS3 may represent substantial selective
benefits for clone 2 to predominate. Analysis of genomic aberrations for this sample (+4q,
+11q, -13q) was not informative as all 42 filtered-tumor cells in the combined study had all
the aberrations (data not shown). Due to the low number of cells comprising clone 1, copy
number aberration calling for these cells was not reliable (data not shown).
Supplementary Figure S5. Tumor evolutionary pattern with two KRAS mutations
acquired in a linear stepwise process. Study of case 90879 reveals: (a) Thumbnail
heatmap of one Fluidigm array used for this analysis. Layout is as in Figure 2. Zoomed
images of each clonal pattern is seen on the right. Note KRAS c.183A>C is firstly acquired
(in clone 2) and then KRAS c.199A>C (clone 3) (b) Hierarchical clustering of 101 tumor cells
shows three clones. Grey, absence; red, mutation/SNP. (c) Clonal phylogeny using only
mutation information demonstrates a linear evolution with stepwise acquisition of mutations.
Note clone 3 is the predominant subclonal population and that the double hit in KRAS is
sequentially acquired. (d) Histograms for the weighted means of the calculated copy number
(wCNV) values for 1q/5p/8p in all sorted tumor single-cells. Red columns mean cells
displaying loss (wCNV≤1.5); grey, no change (1.5<wCNV<2.5); blue, gains (wCNV≥2.5).
Note the higher frequency for genomic aberrations in the tumor as compared with the
reference cell histograms. (e) Clonal phylogeny considering genomic aberrations and
mutations fails to show a clear pattern. Copy number aberration definition and subsequent
filtering approaches have not been as accurate as to define the most plausible clonal
phylogeny. Clonal frequencies are depicted in absolute numbers and percentages.
20
Supplementary Figure S6. Branching evolutionary pattern in case 90482 using only
mutation information. (a) Hierarchical clustering using sorted and filtered cells for mutation
calling (173 cells). Grey means absence, red means presence of mutation or SNP. Note
three tumor clones can be noted. Note clone 2 and 3 have mutually exclusive pattern of
mutations for STK24, ACAD10, and NRAS. (b) Branching evolutionary phylogeny with the
earliest ancestor clone, which has PCDH15 and TRPA mutations (clone 1), giving rise to two
divergent clones: clone 2 (mutant for STK24) and clone 3 (mutant for ACAD10 and NRAS).
Asterisks mean PCHD15 c.2272_2273delinsTA. Clonal frequencies are depicted in absolute
numbers and percentages. (c) Histograms for the weighted means of the calculated copy
number values (wCNV) for 13q in sorted tumor single-cells. Red columns mean cells
displaying loss (wCNV<1.5); grey, no change (1.5<wCNV<2.5); blue, gain (wCNV>2.5). Note
the higher frequency for 13q monosomy in the tumor as compared with the reference cell
histogram. The combination of wCNV data and mutational profiles allows depicting a
comprehensive case phylogenetic history (see Figure 3c).
Supplementary Figure S7. Clonal architecture shifts during patient treatment and
mouse engrafted-myeloma. Phylogenetic natural history of presentation-relapse PCL
patient. (a) Kernel-density plots for the four analyzed samples demonstrate changes in
mutation frequencies. (b) Venn diagram displaying the number of shared and specific
mutations; N/A, not analysed. A selection of genes is shown. (c) Representation of clonal
tides at presentation versus xenograft-1 (left), xenograft-2 (middle) or relapse (right). Clonal
lineages have same colors as in Fig. 6b-d. Note extinction of lineages 2 (yellow), 4 (pink)
and 7 (dark green) during population bottlenecks caused by mouse-engraftment or patienttreatment. Note emergence of two new clones at relapse: lineage 3 (orange) derived from
the earliest ancestor 1 (light-green), and lineage 6 (grey) originated from lineage 3.
21
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23