1075365 pp gene_read_dn_aseqngs_0413_lr


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1075365 pp gene_read_dn_aseqngs_0413_lr

  1. 1. ProductProfile QIAGEN GeneRead DNAseq System for NGS For PCR-enabled target enrichment and library quantification The QIAGEN GeneRead DNAseq System enables focused amplification of genes, and subsequent library quantification with integrated controls that assess sample quality, for next-generation sequencing (NGS). Using multiplex PCR-based target enrichment, the QIAGEN GeneRead DNAseq Gene Panels increase the efficiency of sequencing efforts by focusing on the genes most relevant to a specific disease or research area. Integrated within the panels are control amplification primers that target static regions within the human genome. The QIAGEN GeneRead DNAseq Library Quantification System is a simple, real-time PCR method for library quantification, which also serves as a quality-control checkpoint to assess whether the sample library can be effectively sequenced. The QIAGEN GeneRead DNAseq System for NGS provides: JJ Enrichment JJ Integrated JJ Free to enable ultra-deep sequencing of disease-relevant genes Tumor sample 1 BRAF: K154N controls, enabling quality control before sequencing PDGFRA: S478P TP53: R273C sequence variant analysis software Identification of novel mutations with clear results KRAS: G12V With the growing accessibility of NGS platforms, simplified DNA mutation identification methods are needed to realize the goal of individually tailored Tumor sample 2 Tumor sample 3 ERBB2: P1197R EGFR: C499Y therapies. Genomic DNA saved from tissue samples for molecular analysis is often stored by formalin fixation and paraffin embedding (FFPE), which can result in DNA degradation. It is estimated that only 1–2% of genetic Figure 1. Variants in tumor samples, as detected by the QIAGEN GeneRead DNAseq Lung Cancer Gene Panel. material from an FFPE sample is of sufficient quality for molecular analysis. The new QIAGEN GeneRead DNAseq System is designed to work with these samples and address these concerns. Here, lung cancer FFPE samples Table 1. Identification of low frequency variants in lung adenocarcinoma FFPE samples were assessed for mutations in 20 genes most often mutated in lung cancers Variant frequency (%) KRAS c.35G>T 38 p.G12V c.462A>C 15 p.K154N PDGFRA c.1432T>C 54 p.S478P TP53 c.817C>T 24 p.R273C ERBB2 c.3590C>G 9 p.P1197R KRAS c.35G>T 30 p.G12V EGFR c.1496G>A 7 p.C499Y KRAS c.35G>T 23 p.G12V PDGFRA c.1432T>C 59 p.S478P TP53 Several mutations were present in these samples, with a shared mutation Codon change BRAF using the QIAGEN GeneRead DNAseq Lung Cancer Panel (Table 1). c.817C>T 11 p.R273C Tumor sample Gene 1 among the 3 samples (Figure 1). 2 3 AA change Sample & Assay Technologies
  2. 2. QIAGEN GeneRead DNAseq Gene Panels Gene of interest Focused sequencing for the most important genes DNA sequencing is a useful tool to detect genetic variations, including variants important in diseases like cancer, neurological disease, heritable diseases, developmental disorders, and many others. The QIAGEN GeneRead DNAseq System uses overlapping primer pairs (Figure 2) and a simple workflow (Figure 3) to achieve targeted enrichment of the most important genes in your area of research, and is compatible with a variety of NGS platforms, including the Ion PGM Sequencer, Ion Proton, Figure 2. PCR-enabled targeted enrichment of genes of interest (GOI). The QIAGEN GeneRead DNAseq System employs overlapping primer sets across the exonic portions of a gene or genes, maximizing target coverage and minimizing nonspecific amplification. and Illumina HiSeq and MiSeq sequencing systems. Performance specifications of QIAGEN GeneRead DNAseq Gene Panels Central to the ability to identify point mutations within genomic sequences is the ability of the design strategy to achieve high coverage. This includes, and the values shown (Table 2) indicate: Isolate DNA JJ Design coverage: 90% JJ Amplicon Gene of interest Targeted enrichment (PCR enabled) specificity: 95% JJ Sequence coverage uniformity: 90% In addition, for full confidence in mutation calls, it is recommended that each base be covered at 500x or above. While newer NGS platforms can perform the desirable function of processing multiple samples simultaneously, it is important to keep in mind that the limitations for Library preparation sample processing are determined by the NGS platform. Start adaptor End adaptor Table 2. Multiplex capacity of QIAGEN GeneRead DNAseq Gene Panels in NGS platforms Library quantification Sequencing AT G C AT G Platform AT G G AT TA A C Typical number of reads A A C G TAT G Recommended multiplex level per run for 500x coverage depth 4-gene panel 20-gene panel 124-gene panel PGM 314 > 0.1 million 1 sample X X PGM 316 > 1 million 10 samples 2 samples X PGM 318 > 2 million 20 samples 4 samples 1 sample MiSeq ~ 5 million 40 samples 8 samples 2 samples AT G C AT TAT T T Guided assembly AT G C AT G G AT TA A C G TAT G C AT TAT T T Sequence analysis AT G C AT G G AT TA A C G TAT G C AT TAT T T AT G C AT G G T T TA A C G TAT G C AT TAT T T AT G C AT G G AT TA A C C A G T G C AT TAT T T Wild-type Sample 1 Sample 2 Figure 3. QIAGEN GeneRead DNAseq Gene Panel System targeted enrichment NGS workflow. Extract DNA, and use QIAGEN GeneRead DNAseq Gene Panels for targeted exon enrichment. Then construct your NGS library, quantify and quality-control using the QIAGEN GeneRead Library Quantification System, and perform NGS and data analysis using the QIAGEN NGS Data Analysis Web Portal. www.qiagen.com
  3. 3. QIAGEN GeneRead DNAseq Library Quant Array Accurate quantification and quality control Current methods for sample NGS library quantification all have significant drawbacks, from false positives to a lengthy time-to-result. The new QIAGEN GeneRead DNAseq Library Quant Array serves two purposes: first, to provide a simple library quantification method, and second, to provide a method to assess sample library quality. Completing NGS analysis with multiple samples requires a uniform starting concentration. The QIAGEN GeneRead DNAseq Library Quant Array provides a set of prediluted standards (5 sequential 10-fold dilutions) prealiquoted onto a PCR array plate. The experimental sample library is then assessed against the standard curve (Figure 4). Target DNA PCR primer set (forward and reverse) for target DNA Control DNA PCR primer set (forward and reverse) for control DNA CT Standards (serial dilution) Figure 5. Integrated controls for target enrichment success. QIAGEN GeneRead DNAseq Gene Panels include control regions that can be assessed with the QIAGEN GeneRead DNAseq Library Quant Array. Log (concentration) Your sample library Figure 4. Principle of the QIAGEN GeneRead DNAseq Library Quant Array. The serial dilutions of the DNA standard (5 sequential 10-fold dilutions) generate a standard curve. The sample library should fall within the standard curve. Sample library quality assessment While the ability to selectively target genes of interest decreases costs, NGS is still not an inexpensive tool. Therefore, assessing sample quality prior to NGS analysis is ideal. The QIAGEN GeneRead DNAseq Library Quant Array accomplishes this by measuring integrated controls in the target enrichment process. These controls are primer sets targeted to nonvariable regions of genomic DNA amplified during target enrichment (Figure 5). In addition, by measuring the relative amplification of these controls versus the targets, a QC score is provided that describes the quality of the sample library prior to NGS analysis (Table 3). Table 3. QC score indicates DNASeq library quality (affected by sample quality, target enrichment process, and library construction process). Sample QC score On-target reads (specificity) FFPE-1 (20-gene panel) FFPE-2 (4-gene panel) 1.3 15.2 91% 45% Average read length (bp) 128 bp 24 bp Median coverage depth 206x 83 101 Number of variants per kb region 887x Total number of variants identified 0.79 7.4 Sample Assay Technologies Two FFPE samples were subjected to target enrichment, QC score generation, and NGS. FFPE-1, enriched with a 20-gene panel, showed a QC score of 1.3, while FFPE-2, enriched with a 4-gene panel, had a QC score of 15.2. A QC score from 1–4 indicates high quality, while a score 8 indicates low quality. FFPE-1 showed high specificity, expected average read length, and good coverage depth, while FFPE-2 showed poorer results in all categories, verifying the accuracy of the QC score.
  4. 4. A complete, integrated workflow for NGS analysis Targeted enrichment using the QIAGEN GeneRead DNAseq Gene Panels and subsequent library quantification and quality assessment using the QIAGEN GeneRead DNAseq Library Quant Array are part of a total workflow yielding accurate and trustworthy NGS results (Figure 6). Using the sensitive mutation identification capability of NGS technology, researchers can start to dissect diseases and disorders at a truly individual level. When generated by a system that enriches the most relevant genes in disease and ensures the reliability of sample libraries, NGS results are more focused, more accurate, and more likely to unlock the secrets of cancer and other diseases. DNA extraction Target enrichment Sample pooling/ purification Library preparation Library quantification NGS analysis Sequence analysis Figure 6. QIAGEN’s total workflow for NGS. QIAGEN provides solutions at every stage of preparation for next-generation sequencing, including QIAamp kits for DNA extraction, the GeneRead DNAseq Gene Panels for disease-focused target enrichment, GeneRead Library Prep Kits for library construction, and the GeneRead Library Quantification System, including the GeneRead DNAseq Library Quant Array, for library quantification. Following the NGS run, the complementary GeneRead SeqVariant Analysis software provides fully annotated variant analysis, and qBiomarker Somatic Mutation PCR Arrays provide followup mutation analysis. Product Contents Cat. no. GeneRead DNAseq Library Quant Array Two arrays in formats A, C, D, F, or R, or 1 array in format E or G for GeneRead DNAseq library quantification and quality control of target enrichment process 180601 GeneRead DNAseq Gene Panels Wet-bench verified (cataloged) or bioinformatically verified (custom) primer sets for targeted exon enrichment of a pathway-focused or custom panel of genes 180941 GeneRead Panel Mastermix Mastermix for use with the GeneRead DNAseq Gene Panel System Varies* GeneRead qPCR SYBR Green Mastermix Mastermix for use with GeneRead Library Quant Arrays Varies * Visit www.sabiosciences.com/NGS.php for more information on these products. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Discover more, visit www.qiagen.com/Products/A-Z-List/GeneRead-DNAseq-Gene-Panels! Trademarks: QIAGEN® (QIAGEN Group); Illumina® (Illumina, Inc.); Ion Torrent™, SYBR® (Life Technologies Corp.) 1075365 04/2013 © 2013 QIAGEN, all rights reserved. Australia n 1-800-243-800 Austria n 0800-281011 Belgium n 0800-79612 Brazil n 0800-557779 Canada n 800-572-9613 China n 800-988-0325 Denmark n 80-885945 Finland n 0800-914416 France n 01-60-920-930 Germany n 02103-29-12000 Hong Kong n 800 933 965 India n 1-800-102-4114 Ireland n 1800 555 049 Italy n 800-787980 Japan n 03-6890-7300 Korea (South) n 080-000-7145 Luxembourg n 8002 2076 Mexico n 01-800-7742-436 The Netherlands n 0800-0229592 Norway n 800-18859 Singapore n 1800-742-4368 Spain n 91-630-7050 Sweden n 020-790282 Switzerland n 055-254-22-11 Taiwan n 0080-665-1947 UK n 01293-422-911 USA n 800-426-8157 www.SABiosciences.com www.qiagen.com Sample Assay Technologies