SOMATIC 1 MASTR v2: A flexible NRAS KRAS BRAF panel - TECHNICAL GUIDE MANUFACTURER

Page created by Linda Fowler
 
CONTINUE READING
SOMATIC 1 MASTR v2: A flexible NRAS KRAS BRAF panel - TECHNICAL GUIDE MANUFACTURER
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
SOMATIC 1 MASTR v2: A flexible NRAS KRAS BRAF panel - TECHNICAL GUIDE MANUFACTURER
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

www.multiplicom.com                                                                                          © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                                              Page 2 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

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)

www.multiplicom.com                                                            © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                              Page 3 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

     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.

www.multiplicom.com                                                                 © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                   Page 4 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

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.

www.multiplicom.com                                                              © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                Page 5 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

                            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.

www.multiplicom.com                                                                      © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                        Page 6 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

                                  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)

           www.multiplicom.com                                                                  © 2013 Multiplicom NV, all rights reserved.
           Revision date: January 27, 2014                                                                                    Page 7 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

     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).

www.multiplicom.com                                                              © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                Page 8 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

     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.

www.multiplicom.com                                                                   © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                     Page 9 of 10
TECHNICAL GUIDE: SOMATIC 1 MASTR™ v2

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

www.multiplicom.com                                                                © 2013 Multiplicom NV, all rights reserved.
Revision date: January 27, 2014                                                                                 Page 10 of 10
You can also read