QIAGEN s Preanalytic NGS Solutions

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1 QIAGEN s Preanalytic NGS Solutions For outstanding DNAseq results Choose QIAGEN for every step of your NGS workflow Sample & Assay Technologies

2 1. Next-generation sequencing (NGS) Purification and Sample enrichment Library preparation NGS run Data analysis Next-generation sequencing (NGS): applications and challenges The use of high-throughput NGS technologies continues to provide a wealth of sequence data, resulting in significant advances in a wide range of research areas and applications (Figure 1 A ). Accurate NGS results depend on the ability to rapidly and accurately obtain sequencing data from complex samples, and rely heavily on technologies that provide sufficient yields of targeted, high-quality nucleic acids. QIAGEN, with its leading expertise in sample preparation technologies, has developed a portfolio of dedicated products for your preanalytic DNAseq NGS workflow. High-quality, target-enriched samples and streamlined library preparation procedures with easy-to-use data analysis tools deliver reliable and accurate results, allowing you to overcome the bottlenecks in your NGS workflow and get the most from limited sequencing capacity on any sequencing platform. QIAGEN s NGS solutions have been specifically designed to overcome the numerous challenges present at each step of the workflow, even when working with difficult and limited sample material (Figure 1 B ). A n Cancer research/oncology n Genetic testing n Cardiovascular diseases/cardiology n Gene expression analysis n Genomics n Methylation analysis Research areas n Stem cell research n Plant biology n Evolution n Infectious disease reasearch Applications n Genotyping n Whole genome sequencing n Targeted resequencing n Histone analysis n Microbiology n Metagenomics n Epigenetics n Neurogical disease research B Challenges Simple workflow Time to results and costs Optimal use of sequencing capacity High accuracy with minimal bias Fast turnaround time Easy data analysis Standardization and reproducibility Figure 1. n A NGS applications and n B associated challenges. 2 QIAGEN DNAseq Preanalytic NGS Solutions 05/13

3 2. Purification and Purification and Sample enrichment Library preparation NGS run Data analysis Purification and QIAGEN the gold-standard in DNA purification Even the best and most robust NGS technology can only provide reliable results if the template DNA is of adequate quality and purity. QIAGEN s diverse portfolio of DNA purification technologies is specially adapted to overcome all the challenges of DNA purification, such as low yields and poor quality or challenging sample types (Table 1). For increased standardization and productivity, hands-free preparation of up to 12 samples using proven QIAamp and DNeasy Kits can be automated on the QIAcube (Figure 4). Table 1. Range of DNA purification kits MagAttract HMW DNA Kit Supplier L Supplier B VII M kb Figure 2. Successful isolation of high-molecular-weight DNA. DNA was isolated from 0 µl of blood stabilized with EDTA, citrate, and heparin respectively. Only the MagAttract HMW DNA Kit enabled successful isolation of 0 kb high-molecular-weight DNA. M: Marker. Sample type Plant cells or tissues FFPE tissue Blood Tissues, swabs, CSF, blood, body fluids High-molecular weight DNA Kit DNeasy Plant Mini Kit GeneRead DNA FFPE Kit QIAamp DNA Blood Mini Kit QIAamp DNA Mini Kit MagAttract HMW DNA Kit C T values Purification of high-molecular-weight DNA The MagAttract HMW DNA Kit successfully isolates high-molecularweight (HMW) DNA (100 0 kb) using a simple, magnetic beadbased protocol, and delivers high yields of DNA that is especially suited for NGS applications (Figure 2). For sensitive applications like NGS, it is essential that inhibitors such as anticoagulants, enzymes, and divalent cations are completely eliminated. Real-time PCR of DNA using the MagAttract HMW DNA Kit demonstrates the complete lack of inhibition in all samples (Figure 3) ΔRn µl Citrate-blood 8 µl Citrate-blood 2 µl EDTA-blood 8 µl EDTA-blood 2 µl Heparin-blood 8 µl Heparin-blood 0.3 Figure 3. No inhibition in samples derived from anticoagulated blood. n A and n B Samples were processed with the MagAttract HMW DNA Kit. Different volumes of eluate were used, as indicated. Variable C T values from sample to sample are a result of WBC and content variations Cycle QIAGEN DNAseq Preanalytic NGS Solutions 05/13 Sample & Assay Technologies 3

4 2. Purification and NGS-ready DNA from challenging sample types Figure 4. Automated NGS sample prep with the QIAcube. The innovative QIAcube enables fully automated sample preparation using QIAGEN spin-column kits. Due to fixation and embedding conditions, as well as long-term storage, DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissue often is severely damaged. In particular, the deamination of cytosine bases to deoxy-uracil leads to C T conversions in subsequent sequencing reactions. The GeneRead DNA FFPE Kit provides a streamlined, easy-touse procedure for efficient purification of DNA from FFPE tissue sections that includes removal of deaminated cytosines, thereby preventing false results in DNA-sequencing. Removal of artifacts prior to sequencing enhances precision, enabling lower read coverage that results in valuable time and cost savings. Automate your NGS purification save time while ensuring more standardized results A wide range of QIAGEN kits, including QIAamp Kits, DNeasy Kits, as well as the GeneRead FFPE Kit and GeneRead Size Selection Kit, can be easily automated on the QIAcube. The innovative QIAcube uses advanced technology to process up to 12 QIAGEN spin columns at a time, allowing seamless integration of automated, low-throughput sample prep into your NGS workflow. Sample preparation using the QIAcube follows the same steps as the manual procedure (i.e., bind, wash, and elute) enabling easy purification of high-quality DNA. C T Figure 5. Complete genome coverage. Comprehensive analysis of 267 loci across the entire genome was performed using RT 2 qpcr Primer Assays (QIAGEN) and real-time PCR following DNA with the REPLI-g Single Cell Kit from 3 different single cell experiments. Low and consistent C T values, with no dropout from any marker, indicate that DNA was successfully amplified from all areas of the genome Chromosome number (human) Cell 1 Cell 2 Cell X X 4 QIAGEN DNAseq Preanalytic NGS Solutions 05/13

5 2. Purification and Whole genome from a single cell NGS analyses are often limited by the small amount of sample available. This challenge can be overcome with the REPLI-g Single Cell Kit, which is specially designed to deliver unbiased of genomic DNA from single cells (1 to <1000 bacterial or tumor cells). NGS results comparable to purified genomic DNA, with complete genome coverage, are reliably obtained (see testimonial and Figure 5). A high percentage of sequence coverage and very low error rates are achieved with REPLI-g Single Cell amplified DNA, when starting from a single bacterial cell (Figure 6). Compared to other available single-cell solutions, no marker drop-outs in human single cell analysis can be detected (Figure 7). Compared to our current method, the REPLI-g Single Cell Kit greatly reduced the bias and delivered more uniform whole genome (comparable to non-amplified genomic DNA) of single lymphocytes, making it possible to detect SNPs, CNVs, and SVs simultaneously. No significant differences were observed for nextgeneration sequencing parameters, such as the mean mapping quality, read mapping ratio, and read duplication ratio when compared to the high-quality results obtained using the REPLI-g Mini Kit. Luting Song, Staff Scientist, Oncology Research, Beijing Genome Institute (BGI), China Percent genome covered % REPLI-g Single Cell Kit (1 cell) REPLI-g Single Cell Kit (1 cell) % % REPLI-g Single Cell Kit (1000 cells) gdna REPLI-g Single Cell Kit (1000 cells) gdna Error rate 0.43 % 0.22 % 0.35 % Chimeras 0.08 % 0.06 % 0.11 % Indel rate 0.05 % 0.02 % 0.01 % Figure 6. Comparable NGS results. Whole genome sequencing of the Bacillus subtilis genome was performed on the Illumina MiSeq instrument. For analysis, 2 µg of genomic DNA or DNA amplified from 10 5 cells using the REPLI-g Single Cell Kit was sheared into 300 bp fragments and 1 µg of each was used for library preparation. n A Comparable sequence coverage was observed for gdna and REPLI-g Single Cell amplified DNA. n B Comparison of non-amplified and REPLI-g amplified DNA revealed error rates in a similar, very low, percentage range. C T x x x x REPLI-g Single Cell (QIAGEN) MDA based WGA (Supplier G) PCR based Single Cell WGA (Supplier N) Genomic locus and WGA method PCR based Single Cell WGA (Supplier S) Genomic marker QIAGEN Supplier G Supplier N Supplier S X54 0 % 40 % 40 % 0 % 99 0 % % 0 % 0 % % 0 % 0 % 0 % Figure 7. The REPLI-g Single Cell Kit delivers highly reliable, unbiased of DNA from a single cell. n A The REPLI-g Single Cell Kit or single cell kits from Suppliers G, N, and S were used to individually amplify 5 human cells. Real-time PCR was used to analyze 3 markers to identify loss or variability in the amount of genomic loci. Unlike kits from other suppliers, the REPLI-g Single Cell Kit delivered unbiased of DNA in each of the 5 cells, indicated by equivalent C T values for each marker. n B Unlike with the REPLI-g Single Cell Kit, DNA amplified using the kits from Suppliers G and N demonstrated high dropout rates. For both kits, genomic marker X54 was not amplified in 2 of the 5 cells tested, and the kit from Supplier G did not amplify marker 99 in 1 of the 5 cells, indicating incomplete genome coverage and biased that makes these kits unsuitable for reliable single cell research. QIAGEN DNAseq Preanalytic NGS Solutions 05/13 Sample & Assay Technologies 5

6 3. Sample enrichment Purification and Sample enrichment Library preparation NGS run Data analysis Gene of interest Library quantification A T G C A T G A T G G A T T A A C A A C G T A T G A T G C A T T A T T T Guided assembly A T G C A T G G A T T A A C G T A T G C A T T A T T T Sequence analysis A T G C A T G G A T T A A C G T A T G C A T T A T T T A T G C A T G G T T T A A C G T A T G C A T T A T T T A T G C A T G G A T T A A C C A G T G C A T T A T T T Isolate DNA Targeted enrichment (PCR enabled) Library preparation Start adaptor End adaptor Sequencing Wild-type Sample 1 Sample 2 Figure 8. GeneRead DNAseq Gene Panel System targeted enrichment NGS workflow. Following DNA extraction, the GeneRead DNAseq Gene Panels are used for targeted exon enrichment. Then, the NGS library is constructed and quantified and quality-control using the GeneRead Library Quantification System. Following the NGS run, data analysis is done using the QIAGEN NGS Data Analysis Web Portal. Sample enrichment Focused of your gene(s) of interest Making optimal use of limited sequencing capacity, time, and resources are major challenges in NGS. The GeneRead DNAseq Gene Panel System uses PCR-based targeted exon enrichment for focused of your genes of interest, allowing you to concentrate sequencing efforts on what matters most and save time and money while answering important biological questions (Figure 8). More than 90% of exonic regions are covered by the panel primer design, enabling deep sequencing and the identification of low-frequency genetic variants that might otherwise go undetected (Figure 9 and Table 2). The high specificity of the system maximizes efficient use of sequencing capacity, as more than 85% of sequencing reads align to target regions. High-quality variant calls are ensured with the high-coverage uniformity of the system, with more than 85% of targeted bases covered at >10% median sequencing depth (Table 2). Table 2. Identification of low-frequency variants Sample Gene Codon change Variant frequency (%) AA change Tumor sample 1 KRAS c.35g>t 38 p.g12v BRAF c.462a>c 15 p.k154n PDGFRA c.1432t>c 54 p.s478p TP53 c.817c>t 24 p.r273c Tumor sample 2 ERBB2 c.3590c>g 9 p.p1197r KRAS c.35g>t 30 p.g12v Tumor sample 3 EGFR c.1496g>a 7 p.c499y KRAS c.35g>t 23 p.g12v PDGFRA c.1432t>c 59 p.s478p TP53 c.817c>t 11 p.r273c 6 QIAGEN DNAseq Preanalytic NGS Solutions 05/13

7 3. Sample enrichment Each biology-focused GeneRead DNAseq Gene Panel consists of multiplex PCR primer sets to amplify exonic regions of a thoroughly researched panel of biologically relevant and diseasefocused genes (Table 3). Mix-n-Match and Custom Panels can also be customized to include genes tailored to your specific research interests. Gene of interest Figure 9. PCR-enabled targeted enrichment of genes of interest (GOI). The principle of the GeneRead DNAseq System is to employ overlapping primer sets across the exonic portions of a gene or genes to maximize target coverage and minimize nonspecific. Table 3. GeneRead DNAseq Gene Panels for targeted exon enrichment Targeted Exon Enrichment Panel Biology-focused panel: Cancer Panel ( GOI) Biology-focused panel: High Content Cancer Panel (124 GOI) Mix-n-Match Panels Custom Panels Content Breast cancer Colon cancer Leukemia Liver cancer Lung cancer Ovarian cancer Prostate cancer Gastric cancer Comprehensive cancer panel Create your own panel from 124 genes (all primer sets laboratory-verified) Create your own panel using any gene(s) from the human genome QIAGEN DNAseq Preanalytic NGS Solutions 05/13 Sample & Assay Technologies 7

8 4. Library preparation Purification and Sample enrichment Library preparation NGS run Data analysis Adapter dimers Adapter monomers Figure 10. Precise size selection. The GeneRead Size Selection Kit effectively removes adapter dimers and adapter monomers following library preparation. A scaled-up image of the above data showing the correct size distribution of Illumina-compatible library fragments following size selection is shown in inset. FU: Fluorescence units. Library preparation Optimized, time-saving DNA library preparation GeneRead Library Prep Kits use advanced library preparation technology with an optimized workflow to ensure maximum yields of high-quality library DNA and eliminate the bottlenecks and sample loss that are often major problems in other library preparation methods. The streamlined, one-tube procedure is up to 50% faster than other suppliers (Figure 11). Specific kit formats have been developed that are compatible with Ion Torrent instruments from Life Technologies or HiSeq and MiSeq instruments from Illumina. Highly efficient ligation reactions and an optional, high-fidelity step deliver superior library yields and quality from as low as 50 ng starting material (Figure 12). Following adapter ligation, the GeneRead Size Selection Kit can be used to provide precise, spin-column-based size selection of library DNA, and is automatable on the QIAcube for even more time savings (Figure 10). Supplier I 250 min End repair Cleanup A-addition Adapter ligation Cleanup and size selection HiFi library (optional) Figure 11. Optimized workflow allows more rapid and efficient preparation of Illumina-compatible DNA libraries. The GeneRead Library Prep (I) Kit uses an optimized one-tube protocol, with fewer cleanup steps and optional highfidelity library. The hands-on time and total time required for preparation of library DNA is significantly reduced compared to the library preparation system from Supplier I. QIAGEN 125 min End repair Five tubes Four steps Total time = 150 min Hands-on time = 45 min A-addition Adapter ligation One tube Three steps Total time = 100 min Hands-on time = 5 min Cleanup and size selection HiFi library (optional) 8 QIAGEN DNAseq Preanalytic NGS Solutions 05/13

9 4. Library preparation qpcr-based library quantification Inaccurate quantification of amplifiable library molecules can lead to mixed signals and non-resolvable data or poor yields of template-carrying beads (Ion Torrent platform) or clusters (Illumina platform). The GeneRead DNAseq Library Quant Array provides a set of prediluted standards pre-aliquoted onto a PCR array plate. The experimental sample library is then assessed against the standard curve (Figure 13). A Cumulated base frequency (%) ng Normalized coverage C T Region coverage Standards (serial dilution) Your sample library Figure 13. Principle of 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. The QIAGEN GeneRead DNAseq Library Quant Array assesses sample quality prior to NGS analysis by measuring integrated controls in the target enrichment process (Figure 14). In addition, by measuring the relative of these controls versus the targets, a QC score is provided that describes the quality of the sample library prior to NGS analysis. Log (concentration) B Cumulated base frequency (%) QIAGEN Supplier L Normalized coverage Figure 12. High yields of library DNA with uniform coverage distribution. n A Genomic DNA (50 ng) was made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit. Sequencing using an Illumina MiSeq instrument revealed a median coverage of 49-fold with uniform coverage distribution. n B Genomic DNA was used to generate an Ion Torrent-compatible DNA library. DNA prepared using the GeneRead Library Prep L Kit provided better normalized coverage than DNA prepared using a kit from another supplier. Target DNA PCR primer set (forward and reverse) for target DNA Control DNA PCR primer set (forward and reverse) for control DNA Figure 14. Integrated controls for target enrichment success. QIAGEN GeneRead DNAseq Gene Panels include control regions, which are primer sets targeted to nonvariable regions of genomic DNA amplified during target enrichment that can be assessed with the QIAGEN GeneRead DNAseq Library Quant Array. QIAGEN DNAseq Preanalytic NGS Solutions 05/13 Sample & Assay Technologies 9

10 5. NGS run / 6. Data analysis Purification and Sample enrichment Library preparation NGS run Data analysis NGS run Our complete portfolio of NGS solutions has been designed to deliver the most accurate data possible. Gentle nucleic acid purification, accurate high-fidelity PCR enzymes, and a reduction of PCR cycles minimize the risk for introducing bias and errors. QIAGEN s proven technologies, along with optimized NGS workflows, from purification and through to data analysis, are fully compatible with Illumina MiSeq and HiSeq sequencing platforms, as well as Ion Torrent instruments from Life Technologies. Data analysis New, cloud-based software for GeneRead DNAseq Targeted Exon Enrichment Panels automatically performs all the steps necessary to generate a DNA sequence variant report from your NGS data (Figure 15). Follow just three easy steps to receive a detailed analysis of your NGS data: 1. Data upload: Upload your FASTQ or SFF files into our secure cloud space where analysis takes place. 2. Variant analysis: After selecting a few simple parameters, data analysis begins. Depending on the size of the sequence files, analysis can take from 30 minutes to a few hours to finish. During this step, several processes are taking place in the cloud, including primer clipping, mapping/ alignment, variant calling, variant annotation, and variant reporting. 3. Results download: After the analysis is finished, you will be provided a link to download a summary and detailed variant reports, as well as all the intermediate results. Figure 15. GeneRead targeted exon enrichment panel cloud-based data analysis. This free, easy-touse analysis requires only the GeneRead catalog number, sequencing platform (Ion Torrent PGM or Illumina), analysis mode (germline or somatic), and read mode (single-end or paired-end) to provide detailed analysis of your NGS data QIAGEN DNAseq Preanalytic NGS Solutions 05/13

11 7. Ordering Information Ordering Information Product Contents Cat. no. MagAttract HMW DNA Kit (48) MagAttract Suspension G, buffers, Proteinase K, RNase A DNeasy Plant Mini Kit (50)* Spin columns, RNase A, buffers, collection tubes (2 ml) QIAamp DNA Blood Mini Kit (50)* Spin columns, reagents, buffers, collection tubes (2 ml) GeneRead DNA FFPE Kit (50) Spin columns, reagents, buffers, collection tubes (2 ml) REPLI-g Single Cell Kit (24)* Polymerase, buffers, and reagents for 24 WGA reactions GeneRead DNAseq Gene Panels Wet-bench verified primer sets for targeted exon enrichment GeneRead DNAseq Gene Panels High-Content Wet-bench verified primer sets for targeted exon enrichment GeneRead Custom DNAseq Gene Panels Primer sets for customized targeted exon enrichment GeneRead Mix-n-Match DNAseq Gene Panels GeneRead DNA Library I Core Kit (12) GeneRead DNA I Amp Kit (100) GeneRead Adapter I Set 12-plex (72) GeneRead DNA Library L Core Kit (12) GeneRead DNA L Amp Kit (100) GeneRead Adapter L Set 12-plex (72) Primer sets for customized targeted exon enrichment, selected from QIAGEN s laboratory-verified primer sets Buffers and reagents for end-repair, A-Addition, and ligation, for use with Illumina instruments Buffers and reagents for library, for use with Illumina instruments 12 barcoded adapters for ligation to DNA library, for use with Illumina instruments Buffers and reagents for end-repair, ligation, and nick-repair, for use with Life Technologies instruments Buffers and reagents for library, for use with Life Technologies instruments 12 barcoded adapters for ligation to DNA library, for use with Life Technologies instruments GeneRead DNAseq Library Quant Array PCR arrays and components for library quantification GeneRead Size Selection Kit (50) Spin columns and buffers QIAcube Robotic workstation for automated purification Varies * Larger kit sizes available. 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 or can be requested from QIAGEN Technical Services or your local distributor. Visit and stay up-to-date on innovative NGS solutions! Trademarks: QIAGEN, QIAGEN GeneRead, QIAamp, QIAcube, DNeasy, GeneRead, MagAttract, REPLI-g (QIAGEN Group); Ion Torrent (Life Technologies Corporation); HiSeq, MiSeq (Illumina, Inc.) /13 13 QIAGEN, all rights reserved. QIAGEN DNAseq Preanalytic NGS Solutions 05/13 Sample & Assay Technologies 11

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