Welcome to the NGS webinar series

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1 Welcome to the NGS webinar series Webinar 1 NGS: Introduction to technology, and applications NGS Technology Webinar 2 Targeted NGS for Cancer Research NGS in cancer Webinar 3 NGS: Data analysis for genetic profiling Data analysis pre-set parameters Webinar 4 NGS: Advanced analysis with IVA & CLC bio Cancer Research Workbench Advance data analysis & interpretation

2 Next Generation Sequencing: An Introduction to Applications and Technologies Quan Peng, Ph.D. Scientist, R&D

3 Legal Disclaimers QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. 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. 3

4 Agenda Next Generation Sequencing Background Technologies Applications Workflow Targeted Enrichment Methodology Data analysis Product released! 4

5 DNA Sequencing The Past Decade 10E+8 Output (Kb) 10E+6 10E+4 10E+2 Adapted from ER Mardis. Nature 470, (2011) doi: /nature

6 Rapid Decrease in Cost 6

7 What is Next-Generation Sequencing? Sanger Sequencing NGS: Massive Parallel Sequencing DNA is fragmented.dna is fragmented Adaptors ligated to fragments (Library construction) Cloned to a plasmid vector Cyclic sequencing reaction Clonal amplification of fragments on a solid surface (Bridge PCR or Emulsion PCR) Separation by electrophoresis Readout with fluorescent tags.direct step-by-step detection of each nucleotide base incorporated during the sequencing reaction 7

8 Bridge PCR DNA fragments are flanked with adaptors (Library) A flat surface (Chip) coated with two types of primers, corresponding to the adaptors Amplification proceeds in cycles, with one end of each bridge tethered to the surface Clusters of DNA molecules are generated on the chip. Each cluster is originated from a single DNA fragment Used by Illumina 8

9 Illumina HiSeq/MiSeq Run time 1-10 days Produces Gb of sequence Read length 2X100 bp 2X250bp (paired-end) Cost: $ $0.4/Mb 9

10 Single-end vs. paired-end reading Single-end reading 2 nd strand synthesis Pair-end reading Single-end reading (SE): Sequencer reads a fragment from only one end to the other Paired-end reading (PE): Sequencer reads both ends of the same fragment More sequencing information, reads can be more accurately placed ( mapped ) May not be required for all experiments, more expensive and time-consuming 10

11 Emulsion PCR Fragments, with adaptors, are PCR amplified within a water drop in oil One primer is attached to the surface of a bead DNA molecules are synthesized on the beads. Each bead bears DNA originated from a single DNA fragment Beads with DNA are then deposit into the wells of sequencing chips, one well one bead Used by Roche 454, IonTorrent and SOLiD 11

12 Ion PGM/ Proton Run time 3 hrs Read length bp; homopolymer can be an issue Throughput determined by chip size (ph meter array): 10Mb 5 Gb Cost: $1 - $20/Mb 12

13 Multiplex Sequencing Barcoding Samples Multiple samples with different indices can be combined and put into one sequencing run or into one sequencing lane Depending on the application, we may not need to generate so many reads per sample Save money on sequencing costs (pay per sample) 13

14 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics 14

15 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq Mutation, SNVs, Indels, CNVs, Translocation 15

16 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcripts, Splice variants 16

17 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification 17

18 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq Microbial- Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification Microbial genome Sequence, Microbial ID, Microbiome Sequencing, 18

19 NGS Workflow Sample preparation Isolate samples (DNA/RNA) Qualify and quantify samples Several hours to days Library construction Prepare platform specific library Qualify and quantify library 4-8 hours Sequencing Perform sequencing run reaction on NGS platform 8 hours to several days Data analysis Application specific data analysis pipeline Several hours to days 19

20 QIAGEN s Solution for NGS Workflow Sample preparation GeneRead DNAseq NGS Panel System V2 MagAttract HMW DNA Kit REPLI-g Single Cell Kit GeneRead rrna Depletion Kit GeneRead DNA QuantiMIZE Library construction GeneRead DNA Library Prep Kits GeneRead Size Selection Kit GeneRead Library Quant Kits Sequencing Data analysis GeneRead DNAseq data analysis web portal Ingenuity Variant Analysis Result validation RT 2 Profiler PCR Arrays Somatic Mutation PCR Arrays PyroMark Pyrosequencing CNA/CNV PCR Arrays EpiTect ChIP PCR Arrays 20

21 Agenda Next Generation Sequencing Background Technologies Applications Workflow Targeted Enrichment Methodology Data analysis New product released! 21

22 Targeted Sequencing What is targeted sequencing? Sequencing a sub set of region in the whole-genome Why do we need targeted sequencing? Not all regions in the genome are of interest or relevant to specific study Exome Sequencing: sequencing most of the coding regions of the genome (exome). Protein-coding regions constitute less than 2% of the entire genome Focused panel/hot spot sequencing: focused on the genes or regions of interest What are the advantages of focused panel sequencing? More coverage per sample, more sensitive mutation detection More samples per run, lower cost per sample 22

23 Target Enrichment - Methodology Hybridization capture Large DNA input (1 ug) Long processing time (2-3 days) Large throughput (MB region to whole exome) Sample preparation (DNA isolation) Library construction Hybridization capture (24-72 hrs) Sequencing Data analysis 23

24 Target Enrichment - Methodology Multiplex PCR Small DNA input (< 100ng) Short processing time (several hrs) Relatively small throughput (KB - MB region) Sample preparation (DNA isolation) PCR target enrichment Library construction Sequencing Data analysis 24

25 GeneRead DNAseq Panels V2 Multiplex PCR-based enrichment of gene(s) or genomic region(s) Gene 1 Gene 2 Primer design algorithm PCR Chemistry Primer separation algorithm GeneRead DNAseq V2 GeneRead DNAseq Panel V2 (sets of primer pools/tubes) GeneRead DNAseq PCR Kit V2 Number of pools depends on how far apart the targeted regions are (up to 2500 pairs/pool). Division of gene primer sets into separate pools increases amplification specificity. 25

26 Specifications Experimental performance metrics Application Solid tumors Hematologic malignancies Disease-specific Comprehensive Panel name # genes Target region (bases) Coverage (%) Specificity (%) Uniformity (%) Tumor Actionable Mutations 8 7, Clinically Relevant Tumor 24 39, Myeloid Neoplasms , Breast Cancer , Colorectal Cancer , Liver Cancer , Lung Cancer , Ovarian Cancer , Prostate Cancer , Gastric Cancer , Cardiomyopathy , Carrier Testing , Cancer Predisposition , Comprehensive Cancer , Panel optimization results in outstanding experimental performance metrics 26

27 GeneRead DNAseq Custom Panel 27

28 NGS Data Analysis Base calling From raw data to DNA sequences, generate sequencing reads Mapping to a reference Align the reads to reference sequences Similar to a BLAST search: compares millions of reads against a reference database Variants identification Identify the differences between sample DNA and reference DNA Variant prioritization/filtering/validation/interpretation 28

29 NGS Data Analysis Reference sequence A Sequencing reads alignment C C 29

30 NGS Data Analysis: Sequencing Depth Coverage depth (or depth of coverage): how many times each base has been sequenced or read Unlike Sanger sequencing, in which each sample is sequenced 1-3 times to be confident of its nucleotide identity, NGS generally needs to cover each position many times to make a confident base call, due to relatively high error rate (0.1-1% vs %) Increasing coverage depth is also helpful to identify low frequent mutation in heterogenous samples such as cancer sample Reference sequence NGS reads coverage depth = 4 coverage depth = 2 coverage depth = 3 30

31 NGS Data Analysis: Specificity Specificity: the percentage of sequences that map to the intended targets region of interest number of on-target reads / total number of reads Reference sequence ROI 1 ROI 2 NGS reads Off-target reads On-target reads On-target reads 31

32 NGS Data Analysis: Uniformity Reference sequence Coverage uniformity: measure the evenness of the coverage depth across target region Calculate coverage depth of each position Calculate the median coverage depth Set the lower boundary of the coverage depth relative to median depth (eg. 0.1 X median coverage depth) Calculate the percentage of the target region covered to the depth of or deeper than the lower boundary NGS reads coverage depth = 10 coverage depth = 2 coverage depth = 3 32

33 QIAGEN s Solution Data analysis for the non-bioinformaticians Input Output FASTQ files Panel used Job type Single Matched Tumor/Normal Analysis mode Somatic Germline Bam and VCF files Link to IVA (automatic upload of files) Sequencing metrics Variants detected Copy number alterations Simple and easy-to-use data analysis, at no cost 33

34 Summary Run Summary Specificity Coverage Uniformity Numbers of SNPs and Indels Summary By Gene Specificity Coverage Uniformity # of SNPs and Indels 34

35 Features of Variant Report SNP detection Indel detection 35

36 QIAGEN s GeneRead DNAseq Panel System V2 FOCUS ON YOUR RELEVANT GENES Focused: Biologically relevant content selection enables deep sequencing on relevant genes and identification of rare mutations Flexible: Mix and match any gene of interest NGS platform independent: Functionally validated for Ion Torrent, MiSeq/HiSeq Integrated controls: Enabling quality control of prepared library before sequencing Free, complete and easy of use data analysis tool 36

37 GeneRead NGS Solutions Compatible with major sequencing platforms Sample GeneRead DNA QuantiMIZE GeneRead DNAseq Library Quant qbiomarker Somatic Mutation Assays Insight Sample QC Library QC Variant Confirmation Sample Isolation Targeted Enrichment Library Construction NGS Run Data Analysis Biological Insight GeneRead DNA FFPE Kit REPLI-g Single Cell Kit REPLI-g Cell WGA & WTA Kit GeneRead DNAseq Target enrichment panels Expanded Content in V2 (Cancer, Inherited Diseases, Cardiomyopathy) GeneRead DNA Library Core Kit GeneRead DNA Amp Kit GeneRead Size Selection Kit GeneRead data analysis portal CLC-Bio Ingenuity System s Ingenuity Variant Analysis Workflow QC Step Streamlined, standardized, and automated sample-to-insight workflow 37

38 Questions? Thank you for attending today s webinar! Contact QIAGEN Call:

39 Welcome to the NGS webinar series Webinar 1 NGS: Introduction to technology, and applications NGS Technology Webinar 2 Targeted NGS for Cancer Research NGS in cancer Webinar 3 NGS: Data analysis for genetic profiling Data analysis pre-set parameters Webinar 4 NGS: Advanced analysis with IVA & CLC bio Cancer Research Workbench Advance data analysis & interpretation

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