Sequencing Application Note March 2012 Targeted Sequencing of Leukemia-Associated Genes Using 454 Sequencing Systems GS GType TET2/CBL/KRAS and RUNX1 Primer Sets for the GS Junior and GS FLX Systems. Introduction Targeted sequencing is a focused and efficient method for investigating variation in a few selected exons or full genes of interest. 454 Sequencing Systems, including the GS FLX and GS Junior Systems, have proven to be powerful solutions for deep sequencing of PCR amplicons due to their long, highly accurate sequencing reads. The GS GType RUNX1 Primer Set and GS GType TET2/ CBL/KRAS Primer Set are research assays designed to provide researchers with a comprehensive picture of genetic variations in four key human genes - RUNX1, TET2, CBL, and KRAS - with potential impact in human health. These genes are associated with a range of leukemias and myeloid malignancies. The complete solution, which includes primer sets, associated protocols, and dedicated software, is designed to aid in the investigation of genetic variation associated with developmental defects, disease progression, and residual disease. The assays are well suited for use with either the GS Junior or the GS FLX and GS FLX+ Systems using genomic DNA isolated from Peripheral Blood Mononuclear Cells (PBMCs). The assays have been co-developed with and extensively tested at the MLL Munich Leukemia Laboratory (Munich, Germany, www.mll.com). Additionally, the TET2/CBL/KRAS assay is the result of the International RObustness of Next-Generation Sequencing (IRON) study (Leukemia. 2011; 25(12): 1840 8). For life science research only. Not for use in diagnostic procedures.
Assay Design GS GType RUNX1 Primer Set The GS GType RUNX1 Primer Set consists of oligonucleotide PCR primers for the amplification of exons 3-8 of the human RUNX1 gene (Haematologica. 2011; 96(12): 1874 1877). The dried down primers are arrayed on a 96-well plate and each plate contains 12 Multiplex Identifiers (MIDs) suitable for sequencing 12 independent samples in one GS Junior System run or in one 8-regions gasket of a GS FLX System run. The full kit contains four primer plates for processing 48 samples per kit. GS GType TET2/CBL/KRAS Primer Set The GS GType TET2/CBL/KRAS Primer Set consists of oligonucleotide PCR primers for the amplification of exons 3-11 of the human TET2, exons 8-9 of the human CBL, and exons 2-3 of the human KRAS genes. The dried down primers are arrayed on a 96-well plate and each plate contains three MIDs suitable for sequencing three independent samples in one GS Junior System run or in one 8-regions gasket of a GS FLX System run. The full kit contains four plates for a total of 12 samples per kit. Workflow The workflow (Figure 2) is a five step process that begins with amplicon preparation from 30-60 ng/well of DNA extracted from Ficoll-purified Peripheral Blood Mononuclear Cells (PBMCs). DNA can be extracted using the Roche MagNA Pure Compact Nucleic Acid Isolation kit I or another appropriate method. After amplification, all the amplicons and samples from each individual PCR plate are pooled together for a simple, one tube purification and quantitation. To facilitate the preparation of the samples for empcr amplification, Dilution Quantitation Calculators are available on our website: www.454.com/my454. The Calculators are pre-populated with entry fields for the quantitative DNA measurements for each pool. The pooled material is then processed through empcr amplification and sequencing. Step-by-step workflow videos for the amplification through quantitation steps (steps 1-3 in Figure 2) are also available online at www.454.com/my454. Figure 1: GS GType TET2/CBL/KRAS and RUNX1 Primer Sets consist of dried down oligonucleotide PCR primers arrayed on 96-well plates. For the GS GType RUNX1 assay, there are two supported methods: 1) GS FLX or GS FLX+ Systems: load the pooled samples derived from each primer plate in a single region of an 8-region gasket (96 samples per sequencing Run) 2) GS Junior System: load the pooled samples derived from each primer plate in a single sequencing Run (12 samples per sequencing Run) For the GS GType TET2/CBL/KRAS assay, there are also two supported methods: 1) GS FLX or GS FLX+ Systems: load the pooled samples from each plate in a single region of an 8-regions gasket (24 samples per sequencing Run) 2) GS Junior System: load the pooled samples from each plate in a single sequencing Run (3 samples per sequencing Run) Detailed protocols are available on the my454 portal at www.454.com/my454. Both assays are compatible with all 454 Sequencing Systems. The GS GType RUNX1 Primer Set and GS GType TET2/CBL/ KRAS Primer Set are designed to be used independently. However, they can be used together on a single sequencing run in the GS FLX or GS FLX+ Systems. Figure 2: Workflow for the preparation, sequencing, and analysis of amplicons generated with the GS GType RUNX1 Primer Set or the GS GType TET2/CBL/KRAS Primer Set 2 GS GType TET2/CBL/KRAS & RUNX1 Primer Sets
Data Analysis The samples genotypes may be determined using the GS Amplicon Variant Analyzer (AVA) software provided with the 454 Sequencing Systems. As part of the application, 454 Life Sciences has developed a software script (present in the GS GType Assay Software Add-on v2.0) that automatically creates an AVA project with the sequencing reads aligned to the target genes reference sequences. The script, along with conversion tables from nucleotide sequence to amino acid and publicly available tools such as the Ensembl database (www.ensembl.org), provide a comprehensive solution for proceeding from DNA sequence to biological result. The process of characterizing variants with AVA begins with the Variants table (Figure 3A). The Marker variants are signposts to indicate where the coding region of each exon begins and ends (at AVA positions 70 and 180, in the example of KRAS exon 2 shown on Figure 3); as well as the location of the first and last full codons of this exon on this amplicon (codon 1 at AVA positions 70-72 and codon 37 at positions 178-180 in this example). In the example, AVA further reports three variants, 103 G/T, 126 G/T, and 127 A/T, all three in the coding region. The alignment of variants to the reference sequence and a visual representation of each variant type and frequency are displayed on the AVA Global Align tab (Figure 3B). The bottom panel shows the multiple alignment of the sequencing reads to the reference sequence. The top panel is a stacked histogram indicating the frequency of all the variations observed between the selected reads and the reference sequence. In this example the 126 G/T variant is highlighted in blue on the alignment. The small green arrow at the bottom of the histogram points to the bar representing the variant that is highlighted in the alignment. All three KRAS variants in this example can be seen in the alignment and in the histogram. Note that variant 103 G/T is found in a different group of reads than 126 G/T + 127 A/T (as seen in the aligned reads). The frequencies of the variants are shown on the left axis of the histogram (~13% for 103 G/T; ~18% for 126 G/T and 127 A/T) and in the variant table (Figure 3). Conversion tables are available at www.454.com/my454 to facilitate the straightforward interpretation of nucleotide changes by translating them into amino acid changes. A B Figure 3: Screenshots of the GS Amplicon Variant Analyzer software A) AVA Variants table. B) AVA Global Alignment with the same variants. The top panel is a histogram with observed frequency of the variants as compared to the reference sequence. The bottom panel is an alignment of the sequencing reads to the reference sequence. Sequencing Application Note March 2012 3
Data Analysis Cont. Using the conversion table for KRAS exon 2, one can find the AVA reference position of the variants of interest and determine the corresponding codon. The conversion table for KRAS exon 2 is shown in Figure 4A. In this specific example: 103 G/T: GGT g TGT in position 12 corresponding to the mutation G12C 126 G/T: TTG g TTT in position 19 corresponding to the mutation L19F 127 A/T: ACG g TCG in position 20 corresponding to the mutation T20S and compares each variant from each sample to a reference mutation database. This all-in-one software comparison allows the direct identification of frameshift and mismatch mutations for each individual sample. The JSI Medical Systems SeqNext software and a manual describing its use can be found at http://www.jsi-medisys.de. With this information, the mutations can be looked up in a database such as Ensembl to complete the biological annotation of the variation (Figure 4B). As an alternative to AVA and the conversion tables, full analysis may be performed using third party software tools. An example is the SeqNext software package from JSI Medical Systems (Figure 5). This software interfaces with the data output from the 454 Sequencing System software Figure 5: A screenshot from the JSI Medical Systems SeqNext software illustrating the KRAS mutations in exon 2. A B Figure 4: Easily move from nucleotide variations to protein alterations. A) A conversion table. In this example, nucleotides and amino acids from KRAS exon 2 are oriented with respect to the exon and the full gene. B) A screenshot from the Ensembl database showing the codon and amino acid changes variations in KRAS exon 2. From here, one can link to additional information in Ensembl about the variations. 5 GS GType TET2/CBL/KRAS & RUNX1 Primer Sets
Results To illustrate the range of detected variants and demonstrate the limits of detection we sequenced 14 known leukemia variants in the KRAS, RUNX1 and TET2 genes from 10 PBMC-extracted genomic DNA samples. Samples were independently prepared and sequenced in multiple tests (n=18 to 36) to demonstrate reproducibility. All of the variants were tested by population (Sanger) sequencing to confirm variant content. 454 Sequencing Systems detected the nine variants also detected by Sanger sequencing, as well as an additional five variants below the Sanger sequencing limit of detection (Figure 6). Conclusion The GS GType RUNX1 Primer Set and GS GType TET2/ CBL/KRAS Primer Set in conjunction with the GS GType Assay Software Add-on v2.0 are powerful tools for the investigation of four important genes associated with various types of hematological syndromes and malignancies. These assays are well suited for either the GS Junior, or GS FLX and GS FLX+ Systems, depending upon the desired throughput and number of samples. Figure 6: Distribution of variant frequency results from 10 PBMC-extracted genomic DNA samples with 14 known leukemia variants in the KRAS, RUNX1 and TET2 genes. Samples were independently prepared and sequenced in multiple tests (n=18 to 36) to demonstrate reproducibility. All variants were tested by Sanger sequencing to confirm variant content. 454 Sequencing Systems detected the nine variants also detected by Sanger sequencing, as well as an additional five variants below the Sanger sequencing limit of detection. Sequencing Application Note March 2012 6
References Landscape of TET2 mutations in acute myeloid leukemia. Weissmann S et al. (2011) Leukemia doi: 10.1038/leu.2011.326. TET2 deletions are a recurrent but rare phenomenon in myeloid malignancies and are frequently accompanied by TET2 mutations on the remaining allele. Bacher U et al. (2011) British Journal of Haematology 156(1):67-75. Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia. Grossmann V et al. (2011) Haematologica 96(12):1874-7. The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories. Kohlmann A et al. (2011) Leukemia 25(12):1840-8. Molecular profiling of chronic myelomonocytic leukemia reveals diverse mutations in >80% of patients with TET2 and EZH2 being of high prognostic relevance. Grossmann V et al. (2011) Leukemia 25(5):877-9. A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of cases. Grossmann V et al. (2011) Leukemia 25(3):557-60. Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1. Kohlmann A et al. (2010) J Clin Oncology 28(24): 3858-65. Ordering Information Product Pack Size Cat. No. GS GType TET2/CBL/KRAS Primer Set 1 kit (4 plates) 06 500 498 001 GS GType RUNX1 Primer Set 1 kit (4 plates) 06 500 358 001 View detailed information, references, and more at www.454.com For life science research only. Not for use in diagnostic procedures. RESTRICTION ON USE: Purchaser is only authorized to use 454 Sequencing System Instruments with PicoTiterPlate devices supplied by 454 Life Sciences Corporation and in conformity with the operating procedures contained in the 454 Sequencing System manuals and guides. License Disclaimer information is subject to change or amendment. For current information on license disclaimers for a particular product, please refer to: (https://www.roche-applied-science.com/new/legal/index.jsp?id=legal_000000). 454, 454 LIFE SCIENCES, 454 SEQUENCING, GS FLX, GS GTYPE, GS JUNIOR, EMPCR, PICOTITERPLATE, PTP, and MAGNA PURE are trademarks of Roche.Other brands or product names are trademarks of their respective holders. Published by Roche Diagnostics GmbH Sandhofer Straße 116 68305 Mannheim Germany 2012 Roche Diagnostics. All rights reserved. 06694861001 03/12