Supplementary Information
Additional details on the patient cohorts .......................................................................... 2
Next-generation deep-sequencing target accession numbers (NGS) ............................. 4
PCR primer design for next-generation deep-sequencing............................................... 4
PCR amplification protocols for next-generation deep-sequencing ................................. 6
PCR amplification 96-well plate layout ............................................................................ 7
Assay development phase .............................................................................................. 8
1
Additional details on the patient cohorts
The molecular deep-sequencing RUNX1 mutation analysis was established using a
retrospective pre-characterized proof-of-principle cohort of 24 patients with 105 serial
samples available (Supplementary Figure S1a). Subsequently, starting 07/2010 and
ending 04/2012, an independent 814 AML cases were prospectively investigated as
part of routine diagnostic procedures. For additional serial analyses, 57 AML cases with
detectable RUNX1 mutations were studied at diagnosis and relapse. A prognostic
model was investigated using an independent cohort of 103 AML cases diagnosed
between 08/2005 – 05/2012 with additional samples obtained during course of therapy.
Regarding the overlap of the distinct patient cohorts a Venn diagram is shown in
Supplementary Figure S1b. The prospective cohort of 814 cases did not overlap with
the assay development cohort of 24 cases. However, there were 10 cases overlapping
between the prospective cohort of 814 cases and the 57 patients studied to assess the
stability of RUNX1 mutations between diagnosis and relapse. 44 cases were
overlapping between the prospective cohort of 814 cases and the 103 patients studied
to assess the prognostic impact of residual RUNX1 mutations after treatment with AMLspecific intensive treatment protocols. Nine patients were overlapping across all three
cohorts.
2
(a)
 Stability Model: 57 paired AML samples from diagnosis and relapse
 Prognostic Model: 103 AML cases with clinical follow-up
Assay Development Phase
•
•
•
•
24 patients
18 AML and 6 MDS
105 serial samples
obtained 11/2005 - 04/2010
Prospective Testing
07/2010 – 04/2012 (22 months)
814 AML
cases
at diagnosis
mutational landscape
analysis of RUNX1 mutations
(b)
Supplementary Figure S1
(a) Overview on the distinct patient cohorts during the
assay development, longitudinal analyses, and prospective testing phases for RUNX1
mutation analysis. (b) Venn diagram depicting the overlap between cohorts. A: cohort of
814 prospective AML cases; B: cohort of 57 paired AML samples to assess RUNX1
mutations at diagnosis and relapse; C: cohort of 103 cases to study prognostic impact
of residual RUNX1 mutations.
3
Next-generation deep-sequencing target accession numbers (NGS)
The primer design for sequencing of genomic DNA was based on the Ensembl
accession number given in Supplementary Table S1 (Ensembl 53: Mar 2009).
Supplementary Table S1 Ensembl accession numbers for deep-sequencing
Gene symbol
Ensembl ID
Transcript ID
RUNX1
ENSG00000159216
ENST00000344691
PCR primer design for next-generation deep-sequencing
Supplementary Table S2 lists the corresponding primer pairs for 7 amplicons
representing RUNX1. The median amplicon size was 342 bp (range 341 – 348 bp).
According to the “Guidelines for Amplicon Experimental Design” by the manufacturer,
the 5’-portion was a 25-mer whose sequence is dictated by the requirements of the 454
Sequencing System for binding to the DNA Capture Beads (Lib-A), and for annealing
the emPCR Amplification Primers and the Sequencing Primer (Roche Applied Science).
This 5’-part also ended with the sequencing key “TCAG” used for amplicon sequencing.
We applied two kinds of such primers, termed “Primer A” and “Primer B”, allowing for
the directional sequencing of the target sequence from either end:

primer A fusion sequence: 5' CGT ATC GCC TCC CTC GCG CCA TCA G 3'

primer B fusion sequence: 5' CTA TGC GCC TTG CCA GCC CGC TCA G 3'
Distinct molecular barcode sequences were incorporated into the primer sequences
after the TCAG sequencing key and before the gene-specific sequence (Figure S2A).
4
Supplementary Table S2 Information on PCR primer and amplification protocols
Gene
Strand
Exon
RUNX1
-
RUNX1
Amplicon
1
Forward Sequence 5' -> 3'
GCTGTTTGCAGGGTCCTAAC
Reverse Sequence 5' -> 3'
GGCCTCCGCCTGTCCTC
Length
348
-
2
CATTGCTATTCCTCTGCAACC
GTTTGTTGCCATGAAACGTG
342
RUNX1
-
3
AAATTCCGGGAGTGTTGTCA
GAAAGGTTGAACCCAAGGAA
341
RUNX1
-
4
TGATCTCTTCCCTCCCTCCT
CAGTTGGTCTGGGAAGGTGT
348
ATTTGAACAAGGGCCACTCA
AATGTTCTGCCAACTCCTTCA
342
RUNX1
-
5
RUNX1
-
6
6.01
CTCCGCAACCTCCTACTCAC
CCCACCATGGAGAACTGGTA
342
RUNX1
-
6
6.01
CCCGTTCCAAGCCAGCTC
GCTTGTCGCGAACAGGAG
342
Notes:
Exon, referring to Transcript ID ENST00000344691 the exons are numbered accordingly 1 to 6. However, historically the
numbering could also be exons 3 to 8.
Amplicon, ascending order of PCR products that are overlapping in an exon
Length, base pairs including the sequence-specific primer
5
PCR amplification protocols for next-generation deep-sequencing
PCR protocols were performed with ~40 ng template genomic DNA. All PCR master
mixes were prepared according to the manufacturer’s recommendations using the
FastStart High Fidelity PCR Kit for exons 2, 3, 4, and 5, and using the GC-RICH PCR
System kit for GC-rich exons 1 and 6 (Roche Applied Science, Mannheim, Germany).
PCR reactions were performed using a 96-Well GeneAmp® PCR System 2720 or 9700
instrument (Applied Biosystems, Foster City, CA) including a final cooling step at 4°C
after final elongation (Supplementary Table S3).
Supplementary Table S3 PCR amplification protocol
Step
Cycles
Time (min)
Temperature
Cycles
Time (min)
Temperature
Initial Denaturation
1
5:00
95.0°C
1
5:00
95.0°C
Denaturation
Annealing
Elongation
10
0:30
0:30
0:30
95.0°C
63.0°C  58.0°C
72.0°C
25
0:30
0:30
0:30
95.0°C
58.0°C
72.0°C
1
7:00
72.0°C
Final Elongation
6
PCR amplification 96-well plate layout
Preconfigured GS GType RUNX1 Primer Set 96-well plates allowed single-plex parallel
PCR reactions of up to 12 patients including 12 different MIDs (Roche Applied Science).
After purification and quantification the resulting amplicon products were pooled and the
pools subsequently diluted to a concentration of 1 x 109 molecules/µl (Supplementary
Figure S2B).
Figure S1
A
96-well plate format with pre-configured primers (MID)
B
12 Patients / plate
1
A
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
RUNX1_TM1_E04
RUNX1_TM2_E04
RUNX1_TM3_E04
RUNX1_TM4_E04
RUNX1_TM5_E04
RUNX1_TM6_E04
RUNX1_TM8_E04
RUNX1_TM10_E04
RUNX1_TM13_E04
RUNX1_TM14_E04
RUNX1_TM15_E04
RUNX1_TM16_E04
FastStart
RUNX1_TM1_E05
RUNX1_TM2_E05
RUNX1_TM3_E05
RUNX1_TM4_E05
RUNX1_TM5_E05
RUNX1_TM6_E05
RUNX1_TM8_E05
RUNX1_TM10_E05
RUNX1_TM13_E05
RUNX1_TM14_E05
RUNX1_TM15_E05
RUNX1_TM16_E05
FastStart
RUNX1_TM1_E06
RUNX1_TM2_E06
RUNX1_TM3_E06
RUNX1_TM4_E06
RUNX1_TM5_E06
RUNX1_TM6_E06
RUNX1_TM8_E06
RUNX1_TM10_E06
RUNX1_TM13_E06
RUNX1_TM14_E06
RUNX1_TM15_E06
RUNX1_TM16_E06
FastStart
RUNX1_TM1_E07
RUNX1_TM2_E07
RUNX1_TM3_E07
RUNX1_TM4_E07
RUNX1_TM5_E07
RUNX1_TM6_E07
RUNX1_TM8_E07
RUNX1_TM10_E07
RUNX1_TM13_E07
RUNX1_TM14_E07
RUNX1_TM15_E07
RUNX1_TM16_E07
RUNX1_TM1_E03
RUNX1_TM2_E03
RUNX1_TM3_E03
RUNX1_TM4_E03
RUNX1_TM5_E03
RUNX1_TM6_E03
RUNX1_TM8_E03
RUNX1_TM10_E03
RUNX1_TM13_E03
RUNX1_TM14_E03
RUNX1_TM15_E03
RUNX1_TM16_E03
GC-rich
RUNX1_TM1_E08.1
RUNX1_TM2_E08.1
RUNX1_TM3_E08.1
RUNX1_TM4_E08.1
RUNX1_TM5_E08.1
RUNX1_TM6_E08.1
RUNX1_TM8_E08.1
RUNX1_TM10_E08.1
RUNX1_TM13_E08.1
RUNX1_TM14_E08.1
RUNX1_TM15_E08.1
RUNX1_TM16_E08.1
GC-rich
RUNX1_TM1_E08.2
RUNX1_TM2_E08.2
RUNX1_TM3_E08.2
RUNX1_TM4_E08.2
RUNX1_TM5_E08.2
RUNX1_TM6_E08.2
RUNX1_TM8_E08.2
RUNX1_TM10_E08.2
RUNX1_TM13_E08.2
RUNX1_TM14_E08.2
RUNX1_TM15_E08.2
RUNX1_TM16_E08.2
RUNX1_TM14_E07
RUNX1_TM14_E08.2
MID 1
MID 2
MID 3
MID 4
MID 5
MID 6
MID 8
MID 10
MID 13
MID 14
MID 15
MID 16
FastStart
GC-rich
control
pool of amplicons
Supplementary Figure S2
Amplicon and library preparation. (A) Principle of a
fusion primer setup for 454 amplicon deep-sequencing. Multiplex Identifiers (MIDs) as
provided by the manufacturer were used to assign a molecular barcode to the
amplicons and samples. (B) Layout of the preconfigured RUNX1 primer plate. After
PCR amplification, individual amplicons were purified and pooled.
7
Assay development phase
The RUNX1 next-generation amplicon deep-sequencing assay had been developed
using a pre-characterized cohort of 24 patients (18 AML and 6 MDS cases), diagnosed
between 11/2005 and 04/2010). For each of these patients, one or more molecular
RUNX1 mutations were known from standard routine testing using a combination of
denaturing high-performance liquid chromatography (DHPLC) and direct capillary
Sanger sequencing. This assay development cohort was planned as longitudinal
analysis, i.e. samples were analyzed starting at the time point of diagnosis and also
included serially collected specimens following the course of treatment. In total, 105
samples from peripheral blood (n=19) or bone marrow specimens (n=86) were collected
and analyzed in parallel by DHPLC/direct capillary Sanger sequencing and nextgeneration amplicon deep-sequencing. The 24 patients were investigated with high
sequencing coverage, i.e. in median 3,498 reads per amplicon were generated.
As highlighted in Supplementary Data Spreadsheet 2 a total of 157 comparisons
were made across 105 time points collected for the 24 patients: (i) the NGS analyses
concordantly detected the mutations known from previous testing with conventional
Sanger sequencing method in 53/53 (100%) comparisons. (ii) in 80/80 (100%)
comparisons both methods consistently did not detect any RUNX1 mutation. (iii)
Importantly, in the remaining 21 comparisons (13.4%) a discrepant result was observed.
In all 21/157 comparisons low-level variants were detected using the amplicon deepsequencing assay. In these patients residual clones still were detectable after treatment
had been initiated or NGS detected a change in mutation load earlier than using
conventional methods. As highlighted in Supplementary Figure S3, deep-sequencing
also allowed to closely monitoring the composition of distinct subclones and their
clonality during course of disease. Of note, three comparisons were not performed since
in these analyses only the next-generation sequencing assay had provided results while
the DHPLC/direct capillary Sanger sequencing workflow had failed to produce results.
8
Supplemental Figure S3 RUNX1 Assay development phase. Early detection of an
increasing clone size by deep-sequencing. In this exemplary case (patient #24;
Supplementary Data Spreadsheet 2), over the course of disease 4 distinct variants in
RUNX1 were detectable. Across the 6 time points that were investigated each of the
four variants is given. The deep-sequencing assay allows monitoring a changing
composition of clones. On the right y-axis, the number of reads is indicated (grey bar
graphs), on the left y-axis, the mutation load, i.e. the percentage of reads harboring the
mutation (red bar graphs) is given, respectively.
9