SOMATIC 1 MASTR v2: A flexible NRAS KRAS BRAF panel - TECHNICAL GUIDE MANUFACTURER
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IFU302
v140524
Research Use Only
TECHNICAL GUIDE
SOMATIC 1 MASTR™ v2:
A flexible NRAS‐KRAS‐BRAF panel
MANUFACTURER:
Multiplicom N.V.
Galileïlaan 18
2845 Niel
Belgium
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2
TABLE OF CONTENTS
1. INTRODUCTION ............................................................................................................................... 3
2. SOMATIC 1 MASTR V2 AS MULTI‐PLEX MASTR OR SINGLE‐PLEX MASTR .......................................... 3
2.1. FLEXIBILITY IN SELECTING GENES OF INTEREST ............................................................................... 3
2.2. MULTIPLEX MASTR ........................................................................................................................... 3
2.3. SINGLE‐PLEX MASTR......................................................................................................................... 3
3. DETERMINATION OF THE POOLING SCHEME (SINGLE‐PLEX ONLY) ................................................... 5
3.1. USING FRAGMENT ANALYSIS DATA ................................................................................................. 5
3.2. USING DNA QUALITY‐BASED MATRIX .............................................................................................. 7
3.3. FINAL EQUATION FOR DEFINING THE POOLING SCHEME ................................................................ 7
4. SPECIFIC GUIDELINES FOR NRAS EXON 03 ........................................................................................ 8
5. SPECIFIC GUIDELINES FOR ANALYSIS OF BRAF_EX18_01 .................................................................. 8
6. LIST OF ABBREVIATIONS .................................................................................................................. 10
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1. INTRODUCTION
The SOMATIC 1 MASTR™ v2 is designed for the targeted amplification of the full coding regions of NRAS,
KRAS and BRAF. As for all Multiplicom’s somatic MASTR assays, the amplicons of the SOMATIC 1 MASTR v2
are shorter than compared to germline MASTR assays (168‐255 bp, versus 150‐420 bp, respectively),
enabling efficient amplification of FFPE‐derived DNA.
2. SOMATIC 1 MASTR V2 AS MULTI‐PLEX MASTR OR SINGLE‐PLEX MASTR
2.1. Flexibility in selecting genes of interest
SOMATIC 1 MASTR v2 is designed to allow customer based gene selection according to the type of
cancer, being either:
Full coding region of NRAS, KRAS and BRAF:
When all three (3) plexes are amplified in parallel, a total of 29 unique targeted amplicons will be
amplified covering the full coding region of the NRAS, KRAS and BRAF. The procedure to follow for
this option is the general workflow for Multiplicom’s MASTRs (see Table 1). Please notice that
exon 15 of BRAF, corresponding to amplicon BRAF_ex15_01, is present in both plex 1 and plex 2.
Full coding region of NRAS, KRAS and exon 15 of BRAF:
In case characterization of variants in the full coding region of the BRAF gene is not required,
SOMATIC 1 MASTR v2 offers the possibility of amplifying the full coding region of only NRAS, KRAS
and exon 15 of BRAF in just one plex (i.e plex 1). When running only plex 1, SOMATIC 1 MASTR v2 is
to be used as a “single‐plex MASTR” for which specific IFUs are available for Part II (see Table 1).
Full coding region of BRAF:
A total of 20 targeted amplicons in plex 2 and 3 cover the full coding region of the BRAF gene.
When analysis of only this gene is required, plex 2 and 3 can be run separately. Here too, the
general IFUs for all MASTRs are applicable (see Table 1).
2.2. Multiplex MASTR
After performing the multiplex PCR and subsequent Universal PCR, the Multiplicom workflow consists
of the following steps:
Performing a quality control of the amplification products based on fragment analysis
Mixing of the MASTR derived amplicons of all plexes per sample, to obtain the amplicon libraries
Purification of these amplicon libraries
Tagging of the amplicons per sample (only in specific case of SRA)
Measuring the concentration of the purified amplicon libraries
Pooling of the purified amplicon libraries per MASTR, to obtain the amplicon pool
Pooling of different amplicon pools for different MASTRs (optional)
Preparing the final sequencing sample (MPS instrument dependent procedure, and according to
the manufacturer’s instructions)
2.3. Single‐plex MASTR
The most obvious difference between the workflow for SOMATIC 1 MASTR v2 as a multiple‐plex MASTR
and as a single‐plex MASTR is that for the latter no mixing of the plexes can be performed. Since the
concentration of the tagged MASTR derived fragments (after completing Universal PCR) of a single plex
is low, it is not practical to determine the concentration of the separate amplicon libraries using
classical methods, such as spectrophotometry and fluorometry. Alternatively, the (relative)
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quantification of these libraries can be done based on the data generated by fragment analyzers (in the
quality control step), such as an ABI capillary sequencer with GeneScan module, or an Agilent
Bioanalyzer (or equivalent).
The workflow for single‐plex MASTRs will be as follows:
Performing a quality control of the amplification based on fragment analysis
Determining of the pooling scheme (section 3 below), and pooling of the amplicon libraries
Purification of the obtained amplicon pool
Measuring the concentration of the purified amplicon pool
Pooling of different amplicon pools for different MASTRs (optional)
Final preparation of the sequencing sample (MPS instrument dependent procedure)
All MASTR (except for single‐plex MASTRs*)
Part I Part II MPS instrument Part III
IFU017
454 Roche IFU020
454 MID
IFU016 IFU018
MiSeq, Illumina IFU021
MASTR MID for Illumina MiSeq
IFU241 Ion PGM, Life
IFU022
MID for Ion PGM System Technologies
* Single‐plex MASTRs
Part I Part II MPS instrument Part III
IFU169
454 Roche IFU020
454 MID for specific single‐plex MASTR
IFU168
MID for Illumina MiSeq MiSeq, Illumina IFU021
for specific single‐plex MASTR
IFU016 IFU242
Ion PGM, Life
MASTR MID for Ion PGM System IFU022
Technologies
for specific single‐plex MASTR
IFU239
Sequencers with
SRA for Short Fragment
short reads
specific single‐ Generation
( 200bp)
plex MASTR
Table 1: Schematic representation of the workflow and the use of the appropriate IFUs.
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3. DETERMINATION OF THE POOLING SCHEME (SINGLE‐PLEX ONLY)
To calculate the pooling scheme for single plex MASTRs, the WAL (Weight of the Amplicon Library) can be
calculated in two different ways (section 3.1. and section 3.2.).
3.1. Using fragment analysis data
Calculation of total peak area (TPA) of the different amplicon libraries as a relative quantification of
these libraries enables defining the pooling scheme. In general, the Weight of the Amplicon Library
(WAL) is defined as:
WAL =
In addition, the Tumor Tissue Content (TTC) must be taken into account (for more information, we refer
to section 9.4 of IFU168 or IFU169): as included in the equation in section 3.3 below.
When using a fragment analyzer other than an ABI capillary sequencer with GeneScan module:
calculate the TPA values according to the manufacturer’s instructions and proceed with section 9.4 of
IFU168 or IFU169.
When using an ABI capillary sequencer with GeneScan module: review the data generated as
described in section 9.3. Total peak areas can be extracted from the .fsa files using MAQ‐S software and
the appropriate Assay Description file (.enc) as follows:
Activate the amplicon libraries which you would like to pool by clicking the “Listbox” button and
checking the box in front of the amplicon libraries you need.
Make sure the bins defined by the Assay Description file are positioned correctly over the peaks, as
only the peak intensity within each bin is considered (Figure 1). A detailed explanation on how to
change bin width and/or position is described below:
o If the position and/or width of a certain bin is not positioned correctly (e.g. Figure 1), the bin
can be moved or resized as follows:
(1) To enter the editing state, double click on the bin that needs to be changed. By default,
MAQ‐S will zoom into the region for more precision.
(2) To move a bin, click and drag with the left mouse button.
(3) To resize a bin, click and drag with the right mouse button.
(4) To leave the editing state, just double click and MAQ‐S will zoom out again.
o Once the bins are in place, it is required to:
(1) Re‐analyze the data by re‐selecting the assay,
(2) Export the assay for future use.
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Wrongly positioned bins Correctly positioned bins
Figure 1. In the left panel, two wrongly positioned bins are depicted: the left bin
is too narrow to cover the whole peak area, and the right bin is positioned too
far to the right. By changing the width of the bin and/or position of the bin, a
correct position for each bin can be obtained.
Adjust parameters in the “Export” tab of the “Settings” window as depicted in the figure below:
Click “Apply”
Go to the “Export” button, choose “All active reads (txt)”. Press “Export” and save the file
containing the peak area information.
Open the .txt file with Excel: the rows represent the different amplicon libraries; the columns
represent the different bins on the chromatogram (Figure 2).
To obtain the TPA value per amplicon library: make the total sum of the peak areas of the different
bins per amplicon library.
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Bin name Peak area
Sum of
peak areas
Amplicon per
library amplicon
library
Sum of
total peak
Figure 2. Example of file containing information on the peak area information per amplicon library. areas (TPA)
Column M needs to be added manually. of all
amplicon
libraries
Remarks:
(1) In case the fluorescent signal exceeds the limit of the ABI capillary sequencer (typically showing
peaks with deformed tips), the calculated peak areas are unreliable. In this case, repeat the
fragment analysis starting from a 100 times diluted labeled PCR product.
(2) For the calculation of the TPA, use bins 1_1 to 1_9.
3.2. Using DNA quality‐based matrix
We provide a DNA quality‐based matrix, which can be used to determine the pooling scheme (Table 2).
This matrix is based on Delta Ct values obtained with the Illumina FFPE QC assay, specifying the relation
between sample quality and the WAL.
Notice that also the tumor tissue content (TTC) should be taken into account (for more information, we
refer to section 9.4 of IFU168 or IFU169): as indicated in equation under section 3.3 below.
Delta Ct count WAL
0 ‐ 1.2 1
1.2 – 2.6 5
2.6 – 3.6 10
Table 2. DNA quality‐based matrix
3.3. Final equation for defining the pooling scheme
Calculate the volume of each amplicon library using:
Amplicon library volume [µl] = ∑
With: Volume: end volume of the amplicon pool
WAL: Weight of the Amplicon Library
TTC: tumor tissue content
(e.g., 0.5 for FFPE sample with 50% tumor cells,
1 for FFPE sample with 100% tumor cells OR for genomic DNA)
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Remarks:
(1) For the WAL values, we refer to either section 3.1 or 3.2.
(2) For more detailed information on the TTC factor, review section 9.4 of IFU168 or IFU169.
(3) The maximum volume per amplicon library cannot exceed 20 µl. In case one or more libraries
would require more than 20 µl, choose a lower end volume and recalculate the Amplicon
library volume.
(4) Preferentially, the minimum end volume of the amplicon pool is 40 µl. In case this would not
be feasible, make sure to recalculate the required volume of Agencourt AMPure XP beads
used in the purification of the amplicon pool accordingly (section 9.5 of IFU168 or IFU169).
Pool the amplicon libraries following the obtained pooling scheme.
Vortex briefly (2‐3 s) and centrifuge at 12,000 x g for 10 s.
Proceed with section 9.5 of IFU168 or IFU169 for the purification of the amplicon pool.
4. SPECIFIC GUIDELINES FOR NRAS EXON 03
Since exon03 of the NRAS gene is immediately flanked by a GC‐rich region, the primer composition of
plex 1 of the SOMATIC 1 MASTR v2 contains 1 forward and two different reverse primers which
provided higher coverage of this exon than either one of the F‐R combinations separately. However, this
results in the generation of two separate amplicons that both completely overspan NRAS_ex03 (Figure
7).
Figure 7. Amplicons fromed for NRAS exon03.
For variant analysis in NRAS_ex03, the sequences generated for the overlapping region between
NRAS_ex03_01_1 and NRAS_ex03_01_2 is to be analysed as one sequence. To calculate coverage of
NRAS_ex03, read counts for both amplicons are to be combined.
5. SPECIFIC GUIDELINES FOR ANALYSIS OF BRAF_EX18_01
The genomic region upstream of exon 18 of BRAF contains a highly repetitive sequence (Figure 8; gene
on minus strand). As a result various software packages might have difficulties correctly aligning the
forward sequence generated for the amplicon BRAF_ex18_01.
Figure 8. Repetitive sequence in intron 17 (covered by amplicon BRAF_ex18_01 of the SOMATIC 1 MASTR v2).
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At low limits of detection (tested for 5%, 3% and 1% variant allele frequency), false positive variant calls
will be reported by analysis software due to this misalignment.
When analyzing for variants in amplicon BRAF_ex18_01, caution must be taken to exclude variants
found at positions >2 bp upstream of exon 18. This can be automated in the JSI SeqNext software by
specifying the auto cut settings in the Region Of Interest tab (Figure 9).
Due to this highly repetitive sequence upstream of BRAF exon18 the Illumina MiSeq chemistry v2 and
v3 was shown to return low quality bases for the forward Read stretching over exon18. For variant
analysis in exon 18, this region should be visually inspected and only information from the reversed
Read should be considered for variant calling to reduce the occurrence of false positive results.
Remark: this setting will then apply to all amplicons analysed.
Figure 9. Auto cut settings in the Region Of Interest tab to exclude variant
calling in positions >2 bp upstream of exons of the genes analysed.
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6. LIST OF ABBREVIATIONS
DNA: Deoxyribonucleic acid
FFPE: Formalin fixed paraffin‐embedded
IFU: Instructions For Use
MASTR: Multiplex Amplification of Specific Target for Resequencing
MID: Molecular Identifier
PCR: Polymerase Chain Reaction
Plex: Set of MASTR derived amplicons
RUO: Research Use Only
SRA: Short Read Amplification
SOF: Short Overlapping Fragment
TPA: Total Peak Area
TTC: Tumor Tissue Content
WAL: Weight of the Amplicon Library
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