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Supplemental material
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3
“Complex molecular rearrangements in childhood acute myelogeneous leukemia with
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translocation t(10;11)(p12;q23) revealed by paired-end mapping” by Sujal Ghosh,
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Christoph Bartenhagen, Vera Okpanyi, Michael Gombert, Vera Binder, Andrea Teigler-
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Schlegel, Jutta Bradtke, Silja Röttgers, Martin Dugas and Arndt Borkhardt
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Material and methods
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Cytogenetics
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Classical cytogenetics (GTG-banding) was performed following standard procedures and
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chromosomes were karyotyped according to ISCN 2009 (1)
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Fluorescence in situ hybridization
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For fluorescence in situ hybridization (FISH) a commercial dual color break apart probe
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specific for the MLL gene located at chromosome 11q23 was used (Vysis LSI MLL Dual
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Color, Break Apart Rearrangement Probe, Abbott, Illinois, USA). Preparation was
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performed according to manufacturer’s instructions. In all cases, chromosomes were
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counterstained with DAPI, and digital imaging, documentation, and analysis of the FISH
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signals were performed on a Zeiss Axioplan 2 fluorescence microscope equipped with
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appropriate filters and an Isis image analysis system (Metasystems, Altlussheim,
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Germany). G-band-like images were achieved by use of the software to convert and
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enhance the gray scale of the DAPI images to black and white. For each sample 100
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nuclei were analyzed and a distance of 3 and more signal diameters was counted as
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splitting.
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Paired-end Sequencing
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DNA was isolated from peripheral blood lymphocytes with DNA AllPrep DNA/RNA/Protein
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Mini Kit (Qiagen, Hilden, Germany).
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NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). 2 – 3
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µg DNA was sheared on a Covaris S2 (Covaris Inc., Woburn, MA, USA). Illumina fragment
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libraries (Paired-End Sample Preparation, Illumina Inc., San Diego, CA, USA) with a
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median insert size of 450 bp were prepared using the SPRI works I method (Beckman-
Quantity and quality were determined on the
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Coulter, Krefeld, Germany) according to both manufacturer’s instructions. Samples of
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patients 1-3 were sequenced on a GAIIx platform; after an in-house upgrade sequencing
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of samples of patients 4-6 sequencing was performed on a HiSeq 2000 (both Illumina Inc.)
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Bioinformatical analysis
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Alignment
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The paired-end alignment against the human reference genome (hg19/GRCh 37) has
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been done with the Burrows-Wheeler Alignment Tool (BWA) (version 0.5.8c) (2) using the
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default settings. The allowed number of mismatches has been set to 2 (GAIIx runs with
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36bp reads) or 3 (HiSeq2000 runs with 50bp reads). The alignment consists of two
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consecutive steps: 1. The global alignment against the reference genome for every end of
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a read pair individually. 2. The assembly of both alignments of every pair with respect to
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their insert size. This may include a local alignment of previously unmapped reads if the
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mate could be mapped properly. All BWA alignments were given in Sequence
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Alignment/Map (SAM) format (3) which has been converted into its binary equivalent, the
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BAM format, for all following analyses.
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Alignment postprocessing
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Duplicated
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MarkDuplicates from the Picard utilities (version 1.46) (http://www.picard.sourceforge.net)
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has been used to remove reads having identical 5' mapping coordinates (both ends of a
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paired-end read) and orientation.
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Sorting of the alignment, either by mapping coordinate or by read name, has been done
57
with Picard as well.
reads
were
excluded
from
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Copy number analysis with sequencing data
all
subsequent
analyses.
The
function
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The program FREEC (version 3.92) (4) was used to estimate copy number variations. It
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takes the paired initial/remission or relapse/remission samples and normalizes the read
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counts across fixed windows of size 10kb by performing a least-squares polynomial fitting
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of read counts in the diseased sample and the corresponding control/remission. The
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following segmentation step used LASSO regression (5) to merge the windows into larger,
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contiguous regions showing copy number gains or losses. For more details on the
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algorithm, see the program's publication (4). Except for the window size, all other
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paramters were set to the default settings. Reads having a mapping quality below 10 were
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excluded before copy number analysis.
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Structural variation (SV) detection with paired-end sequencing data
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Detection of translocations, deletions and inversions, was carried out with GASV(6) based
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on the mapping coordinates (different chromosomes), anomalous insert sizes (greater
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than the mean plus three times the standard deviation) or read orientations (inversions) of
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the read pairs. First, such aberrant read pairs were filtered and compared to the
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control/remission sample. Only uniquely mapped reads having a mapping quality of at
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least 35 were used in the initial and relapse samples. For the control/remission, the quality
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threshold has been set to 5. To correct false positive SVs due to mismappings, aberrant
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read pairs having a proper alternative alignment with BLAT (7) were excluded. Finally,
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those reads left from the intial or relapse sample after filtering and subtraction of the
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remission were then joined together to clusters each representing the same SV
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breakpoint. Only clusters with a minimum size of two (GAIIx runs) or three reads
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(HiSeq2000 runs) and not overlapping SVs listed in the Database of Genomic Variants
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(DGV) [3] were considered for further analysis. For more details on the algorithm for SV
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detection and clustering, see the GASV publication (8). This approach, relying on aberrant
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read pairs, yields approximate regions spanning a few hundred base pairs containing the
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breakpoint (resolution depends on the insert size distribution). The region coordinates and
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associated read orientations were used for primer design for validation by conventional
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PCR. Genomic annotations, like affected genes, pathways and coding regions derived
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from the KEGG (9) and Ensembl databases (10) and breakpoint overlaps between patients
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and sequence depth of neighbouring regions (5kb up- and downstream) of the breakpoint
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regions were computed within R (11) to assist SV selection for subsequent validation.
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Manual inspection of reads
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Figure S1. The Integrative Genomics Viewer (IGV) (Broad Insitute, Cambridge, MA, USA)
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was used for next generation genomic data visualization (12).
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Visualization
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The circular plots were created with Circos (13). The outer ring shows the copy-number
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ratios of 100kb windows as an orange scatter plot while larger segments with copy-number
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gains or losses were highlighted in green and red respectively. All translocations detected,
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according to the filter critria mentioned in the section before, were drawn as black links
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between the affected chromosomes. The copy-number profiles show ratios of 10kb
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windows and were created within R (11).
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Validation
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Selected translocations detected by paired-end read analysis were validated by
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conventional PCR. Detailed information for the primers can be obtained from the
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corresponding author. Capillary sequencing of the products was performed on an Applied
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Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, Ca, USA).
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Results
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Sequencing performance
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Due to upgrade of the sequencing platform, we had a significantly increased output with
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the newer one; nevertheless the defining t(10;11) translocation could be identified with
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both platforms. The GAIIx produced 96,000,000 to 287,000,000 total reads per sample (2-
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6 lanes of one flow cell), the HiSeq2000 201,000,000 to 670,000,000 total reads per
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sample (1-2 lanes of one flow cell). In each sample > 90% of reads could be aligned,
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furthermore fragment coverage (percentage of the genome, which was covered by paired-
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end fragments) was > 90% in each sample. For further coverage details see Table S2. In
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each sample we observed numerous structural variants (translocations, inversions and
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deletions). However, in-house studies with other sequencing projects show that library
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preparation is prone to false-positive variants, which occurred in all in-house sequencing
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projects dealing with biologically distinct malignancies. We established an in-house
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“blacklist” to exclude these variants (can be obtained by the corresponding author).
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Practically, paired-end analysis identifies base sequences of two ends (reads) of a
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previously PCR amplified DNA fragment. The main principle is to acknowledge the fact
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that these two paired-end reads are always base sequences supposed to be orientated
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towards each other encompassing a certain fragment length in-between.
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Alignment leads to the comparison of each read pair with the reference genome. In case of
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alignment of one read to a certain DNA sequence in the reference genome, and alignment
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of the corresponding read (mate) to a DNA sequence, which is not orientated towards its
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mate in a certain distance (fragment length), a structural variant is detected.
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Results patients 1,2,3 and 5
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Patient 1
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Figure S2. Previous FISH and cytogenetic analysis revealed a derivate chromosome 10
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with
der(10)t(10;11)(p12;q23)inv(11q13q23)
and
chromosome
11
with
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der(11)t(10;11)(p12;q13). Sequencing results in this patient showed paired-end reads
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between MLLT10 (m1a) and MLL (m1). Hence, we assume that these reads represented
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the fusion gene consisting of MLL/MLLT10. The breakpoint on 10p12.31 was located in the
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intronic region between exon 8 and 9 of MLLT10. The second breakpoint on 11q23.3 lied
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between exon 10 and 11 of the MLL gene. We identified paired-end reads on chromosome
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11 (m4, m4a), which both lied in the same orientation (minus-strand 11q14.1 and 11q23.3),
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instead of pointing towards each other. This indicates an inversion of the fragment in-
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between. In conclusion, we deduce that after inversion inv(11)(q14.1q23.3), the inverted
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part was translocated to the derivative chromosome 10. Thus, the third breakpoint lied on
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11q14.1. However, the derivative chromosome 11 remains unclear. Reads were found
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around a suspected breakpoint on 16q23.3, which were paired with reads near the
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MLLT10 (m2, m2a) and the 11q13.1 (m3, m3a) breakpoint. These data suggest a
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previously undetected involvement of chromosome 16 in the 10;11 rearrangement, as
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shown in Figure S2C. However, the latter reads were not found in the relapse sample, but
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could be missed due to low coverage. There were no significant large areas of gains or
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losses in copy number (Figure S2F).
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Patient 2
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Figure S3. In cytogenetics only two aberrant metaphases with an inconspicuous
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chromosome 10 and a large metacentric chromosome 11 were revealed. In both
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metaphases chromosome 12 was missing. At least one marker chromosome was found.
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Interphase FISH aimed in 68% of cells with MLL-splitting. Neither in the MLL-FISH
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analysis nor in the M-FISH analysis aberrant metaphases were found.
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Paired-end sequencing revealed the MLL/MLLT10 translocation finding reads (m1a) at the
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3’end of the intronic region 4/5 of MLLT10 and mates (m1) at the 5’end of the intronic
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region 8/9 of the MLL gene. A second mate pair with a low coverage was found; one read
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at the direct 5’end (m2a) of the MLLT10 breakpoint and the mate approximately 2Mb
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upstream the 5’end from the reported MLL breakpoint (m2). However, both reads (m1 and
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m2) were orientated in the same direction. As seen in patient 1 and patient 5, these data
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suggest that an inversion occurred on chromosome 11q23.3. Detection of copy number
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variations showed areas of gains in the long arms of chromosome 1, 13 and 21 and a loss
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in chromosome 12p (Figure S3E). The latter were not found by cytogenetics. These might
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be included in the marker chromosomes.
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Patient 3
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Figure S4. Cytogenetics and FISH deteced an insertion of long arm material of
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chromosome 11 into 10p, but no additional aberrations (46,XY,ins(10;11)(p11;q23q12).
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Reads were located at the 3’end (minus-strand) (m1) and 5’end (plus-strand) (m3) of the
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intronic region 8/9 in the MLLT10 gene (10p12.3). Counterparts were detected in 11q12.1
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(m3a) and 11q23.3 (m1a), respectively, indicating the MLL/MLLT10 fusion gene (Figure
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S4C). Furthermore a “deleted” region within chromosome 11 could be verified (m2, m2a);
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these reads were orientated towards each other correctly but encompass a fragment of
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60Mb, instead of the sequenced fragment size of 450bp only. These reads were located
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on opposite sides of the breakpoints (11q12.1 and 11q23.3). M1/m1a and m3/m3a indicate
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that the genomic fragment, which is “deleted” on 11q, was subsequently inserted into the
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MLLT10 gene. There were no significant changes in copy number (Figure S4E). Validation
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PCR and capillary sequencing verified the MLL/MLLT10 breakpoint (Figure S4D).
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Patient 5
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Figure S5. Cytogenetics and FISH could verify a 46,XX,t(10;11)(p12;q23)inv(11)(q14q23)
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karyotype. Next generation data detected, similar to patient 1, an inversion between
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11q14.2 and 11q23.3, as both mates (m2, m2a) of a read pair, encompassing a 28Mb
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spanning region, were located in the same orientation on the minus strand, instead of
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pointing towards each other.
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Furthermore the MLL/MLLT10 gene fusion was detected by m1/m1a (orientated towards
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the breakpoints in the intronic region of 9-10 of the MLLT10 gene and intronic region 8-9 of
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the MLL gene). Hence, we can deduce from this pattern that after an inversion of the
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indicated region had occurred on chromosome 11, the whole fragment and telomeric
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region was translocated to the MLLT10 region on chromosome 10. M3, m3a indicated the
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reciprocal translocation on the derivative chromosome 11 (q14.2) and the terminal region
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p12.31 of chromosome 10. Validation PCR and capillary sequencing verified the
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MLL/MLLT10 breakpoint, confirming the fusion gene consisting of MLL exon 1-8 and
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MLLT10 exon 9-24 (Fig. S5D). There were no significant changes in copy number.
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References
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1.
Shaffer LG SM, Campbell LJ. ISCN 2009: An International System for Human
Cytogenetic Nomenclature. S. Karger, 2009.
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2.
Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics (Oxford, England) 2009 Jul 15; 25(14): 1754-1760.
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3.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence
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15; 25(16): 2078-2079.
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4.
Boeva V, Zinovyev A, Bleakley K, Vert JP, Janoueix-Lerosey I, Delattre O, et al.
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Control-free calling of copy number alterations in deep-sequencing data using GC-
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content normalization. Bioinformatics (Oxford, England) 2011 Jan 15; 27(2): 268-
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269.
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5.
Tibshirani R. Regression shrinkage and selection via the lasso. J Royal Statist Soc
B 1996; 58(1): 267-288.
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Sindi S, Helman E, Bashir A, Raphael BJ. A geometric approach for classification
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and comparison of structural variants. Bioinformatics (Oxford, England) 2009 Jun
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15; 25(12): i222-230.
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7.
Kent WJ. BLAT--the BLAST-like alignment tool. Genome research 2002 Apr; 12(4):
656-664.
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8.
Zhang J, Feuk L, Duggan GE, Khaja R, Scherer SW. Development of bioinformatics
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resources for display and analysis of copy number and other structural variants in
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the human genome. Cytogenetic and genome research 2006; 115(3-4): 205-214.
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9.
Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and
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interpretation of large-scale molecular data sets. Nucleic acids research 2012 Jan;
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40(Database issue): D109-114.
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Flicek P, Amode MR, Barrell D, Beal K, Brent S, Chen Y, et al. Ensembl 2011.
Nucleic acids research 2011 Jan; 39(Database issue): D800-806.
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11.
R Core Team R. A Language and Environment for Statistical Computing.
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Available from: http://www.R-project.org
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12.
Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV):
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high-performance genomics data visualization and exploration. Briefings in
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bioinformatics 2012 Apr 19.
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13.
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos:
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an information aesthetic for comparative genomics. Genome research 2009 Sep;
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19(9): 1639-1645.
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Tables
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258
patient
no./ sex
1
female
2
female
FAB
3
male
4
female
5
female
6
male
MLL
M4
34%
M5
68%
M4
71,5%
76,50%
M5
51%
M5
88,5%
karyotyping
46,XX,der(10)t(10;11)(p12;q23)inv(11q13q23),
der(11)t(10;11)(p12;q13)
43~45,XX,der(10)?t(10;11)(p11;q23),
der(11)?t(10;11)(p11;q23),2mar,inc[cp2]/46,XX[
7]
46,XY,ins(10;11)(p11;q23q12)
45,XX,-13,der(17)t(13;17)(q31;p13).ish
ins(10;11)(p12;q23q23)(5’MLL+;3’MLL+)
46,XX,t(10;11)(p12;q23)inv(11)(q1?4q23)
46,XY,der(10)t(10;11)(p12;q23)inv(11)(q13q23),
der(11)t(10;11)(p12;q13),-17,+mar[9]/45, idem,Y[2]/46,XY[3]
age at
diagnosis
13 mo.
age at
relapse
17 mo.
18 yrs.
10½ yrs.
5¼ yrs.
5¾ yrs.
9 mo.
24 mo.
8 yrs.
Table S1. Patient characteristics
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260
Patient
261
262
263
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Platform
Initial
Remission
Relapse
lanes
SC
FC
lanes
SC
FC
lanes
SC
FC
3
85%
3x
91%
11,6x
1
GAIIx
6
84%
2,8x
90%
9,6x
2
67%
1,5x
89%
5,5x
2
GAIIx
3
75%
1,8x
90%
7,8x
2
57%
1x
88%
4x
3
GAIIx
3
75%
1,8x
92%
7,8x
2
63%
1,2x
90%
5,1x
4
HiSeq2000
1
91%
5,6x
92%
18,2x
2*
91%
9,52x
92%
30x
2*
91%
8,6X
92%
28x
5
HiSeq2000
1
91%
9,6x
92%
26,9x
1
91%
8,5x
92%
26x
1
91%
9,2x
92%
27x
6
HiSeq2000
1
87%
3x
92%
9,1x
1
92%
6,8x
92%
21,5x
Table S2. Sequencing results: SC = sequence coverage, FC = fragment coverage, *TruSeq v3 used for
library preparation, TruSeq v4 for other samples patient 4 to 6.
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Figure legends
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268
269
270
271
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Figure S1. IGV displays paired-end reads aligned to the reference genome. The light gray bars indicate
reads, in which both mates (~ 450bp distance from each other) can be aligned perfectly to the reference
genome (depicted in the top bar). Colored reads indicate structural variants, their mate pairs appear in the
same color. In case of corresponding mates on different chromosomes a translocation is indicated. If reads
span a region on the same chromosome larger or smaller than the 450bp fragment length an insertion or
deletion, respectively, is revealed. Paired-end reads are supposed to be orientated towards each other. In
case alignment depicts reads in the same orientation, an inversion is indicated.
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274
275
276
277
278
279
280
281
282
283
284
285
Figure S2. patient 1 - A) FISH analysis: Green color of the MLL probe identifies the proximal 5’ part of the
MLL gene, red color identifies the distal 3’part of the gene. The MLL probe shows a MLL split signal (both
green and red) on 10p. B) Karyotyping results C) Rearrangement profile: Molecular pattern of
rearrangement, revealed by paired-end sequencing. The illustration consists of a normal reference genome
in the upper region with found paired-end reads (e.g. m1 and m1a) aligned to the genome. As each read
(e.g. m1) is supposed to be orientated to its mate (e.g. m1a) the type of rearrangement can be deduced; e.g.
m4a and m4 on the same strand suggest an inversion, m1 and m1a on different chromosomes suggest a
translocation D) Schematic overview of the translocation harboring the MLL/MLLT10 fusion gene. E)
CIRCOS plot of the initial sample: Genomic landscape of interchromosomal translocations were scattered
across the whole genome and allocated along the outer ring (chromosome ideograms). The inner ring
represents copy-number status in terms of gains and losses. F) CNV plot: Deep blue colored bars indicate
the copy number determined by sequencing data in relation to the reference genome. In case of gains and
losses bars are elevated or lowered.
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287
288
289
Figure S3. patient 2 - A) Karyotyping results B) Rearrangement profile: Molecular pattern of rearrangement
C) Schematic overview of the translocation harboring the MLL/MLLT10 fusion gene D) CIRCOS plot of the
initial sample E) CNV plot: We detected gains in chromosome 1, 13 and 21 with 3 copies instead of 2 in the
depicted regions. In chromosome 12 we see a loss.
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291
292
293
294
Figure S4. patient 3 - A) FISH analysis: The MLL probe shows a MLL split signal on different chromosomes,
on 10p and 11q respectively, indicating an insertion B) Karyotyping results C) Molecular pattern of
rearrangement D) Schematic overview of the translocation harboring the MLL/MLLT10 fusion gene. The
fusion sequence was further validated by capillary sequencing revealing the breakpoint at 1-bp level. E)
CIRCOS plot of the initial sample F) CNV plot.
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296
297
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Figure S5. patient 5 - A) FISH analysis: The MLL probe shows a MLL split signal (both green and red) on
10p B) Karyotyping results C) Molecular pattern of rearrangement D) Schematic overview of the
translocation harboring the MLL/MLLT10 fusion gene. The fusion sequence was further validated by capillary
sequencing revealing the breakpoint at 1-bp level. E) CIRCOS plot of the initial sample F) CNV plot.
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300
Figure S6. Location of MLL, MLLT10, RNF169 and RNF214 on chromosome 10 and 11 and their
corresponding distances.
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