Next-Generation Sequencing an Intro to Tech and Applications: NGS Tech Overview Webinar Series Part 1
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This slidedeck provides a technical overview of DNA/RNA preprocessing, template preparation, sequencing and data analysis. It covers the applications for NGS technologies, including guidelines for how to select the technology that will best address your biological question.
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Next-Generation Sequencing an Intro to Tech and Applications: NGS Tech Overview Webinar Series Part 1
- 1. Sample to Insight Next Generation Sequencing: An Introduction to Applications and Technologies Quan Peng, Senior Scientist, Genomics Assay Development, Research Foundation Eric Lader, Senior Director, Research and Development 1 Part 1 NGS Intro
- 2. Sample to Insight Legal disclaimer NGS Part 1: Introduction 2 • 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 www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Agenda Next Generation Sequencing • Background • Technologies • Applications • Workflow Targeted Enrichment • Methodologies • Data analysis NGS Part 1: Introduction 3 1 2
- 4. Sample to Insight Agenda Next Generation Sequencing • Background • Technologies • Applications • Workflow Targeted Enrichment • Methodologies • Data analysis NGS Part 1: Introduction 4 1 2
- 5. Sample to Insight DNA Sequencing – Machine Output 10E+2 10E+4 10E+6 10E+8 Output(Kb) Adapted from ER Mardis. Nature 470, 198-203 (2011) NGS Part 1: Introduction 5
- 6. Sample to Insight Rapid decrease in cost NGS Part 1: Introduction 6
- 7. Sample to Insight Almost the $1000 genome…. NGS Part 1: Introduction 7
- 8. Sample to Insight What is Next-Generation Sequencing? DNA is fragmented Cloned to a plasmid vector Or single PCR fragment Cyclic sequencing reaction Separation by electrophoresis Readout with fluorescent tags Automated Sanger Sequencing one residue at a time NGS Part 1: Introduction 8
- 9. Sample to Insight What is Next-Generation Sequencing? DNA is fragmented Cloned to a plasmid vector Or single PCR fragment Cyclic sequencing reaction Separation by electrophoresis Readout with fluorescent tags Automated Sanger Sequencing one residue at a time NGS: Massive Parallel Sequencing .DNA is fragmented Adaptors ligated to fragments (Library construction) Clonal amplification of fragments on a solid surface (Bridge PCR or Emulsion PCR) .Direct step-by-step detection of each nucleotide base incorporated during the sequencing reaction NGS Part 1: Introduction 9
- 10. Sample to Insight Bridge PCR • DNA fragments are flanked with adaptors (Library) • A solid surface is coated with primers complementary to the two adaptor sequences • PCR Amplification, 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, and is thus a clonal population. • Used by Illumina NGS Part 1: Introduction 10
- 11. Sample to Insight Illumina HiSeq/MiSeq • Run time 1- 10 days • Produces 2 - 600 Gb of sequence • Read length 2X100 bp – 2X250bp (paired-end) • Cost: $0.05 - $0.4/Mb NGS Part 1: Introduction 11
- 12. Sample to Insight Illumina Single-End vs. Paired-End • Single-end reading (SE): o Sequencer reads a fragment from only one primer binding site • Paired-end reading (PE): o Sequencer reads both ends of the same fragment o More sequencing information, reads can be more accurately placed (“mapped”) o May not be required for all experiments, more expensive and time-consuming o Required for high-order multiplexing of samples (indexes on both sides) Single-end reading 2nd strand synthesis Pair-end reading NGS Part 1: Introduction 12
- 13. Sample to Insight Emulsion PCR: Ion, Solid, 454 • Fragments with adaptors (the library) are PCR amplified within a water drop in oil • One PCR primer is attached to the surface of a bead • DNA molecules are synthesized on the beads in the water droplet. Each bead bears clonal DNA originated from a single DNA fragment • Beads (with attached DNA) are then deposit into the wells of sequencing chips, one well one bead • Used by Roche 454, IonTorrent and SOLiD NGS Part 1: Introduction 13
- 14. Sample to Insight Ion Torrent PGM/ Proton • Run time 3 hrs – no termination and deprotection steps • Read length 100‐300 bp; homopolymer can be an issue • Throughput determined by chip size (pH meter array): 10Mb – 5 Gb • Cost: $1 - $20/Mb NGS Part 1: Introduction 14
- 15. Sample to Insight Multiplexing samples for sequencing: Sample indexing • Multiple samples with different indices can be combined and sequenced together • Depending on the application, may not need to generate many reads per sample (e.g. SNP) • Save money on sequencing costs (more samples per run), optimize use of read budget NGS Part 1: Introduction 15
- 16. Sample to Insight NGS applications Next Generation Sequencing Genomics Transcript- omics Epi- genomics Meta- genomics NGS Part 1: Introduction 16
- 17. Sample to Insight Next Generation Sequencing Genomics Transcript- omics Epi- genomics Meta- genomics DNA-Seq Mutation, SNVs, Indels, CNVs, Translocation NGS applications NGS Part 1: Introduction 17
- 18. Sample to Insight Next Generation Sequencing Genomics Transcript- omics Epi- genomics Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcripts, Splice variants Meta- genomics DNA-Seq RNA-Seq NGS applications NGS Part 1: Introduction 18
- 19. Sample to Insight Next Generation Sequencing Genomics Transcript- omics Epi- genomics Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification Meta- genomics DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq NGS applications NGS Part 1: Introduction 19
- 20. Sample to Insight Next Generation Sequencing Genomics Transcript- omics Epi- genomics Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification Meta- genomics Microbial genome Sequence, Microbial ID, Microbiome Sequencing DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq Microbial- Seq NGS applications NGS Part 1: Introduction 20
- 21. Sample to Insight Considerations of NGS experiments: Read Budget Read Budget is platform specific and determines multiplexing capacity Read budget requirements differ for different applications Genome Methylome Whole transcriptome Exome Targeted gene expression Fusion transcripts Somatic mutation detection Structural variants Germline SNP Readsneeded Multiplexingpossibility NGS Part 1: Introduction 21
- 22. Sample to Insight NGS workflow Sample preparation • Isolate samples (DNA/RNA) • Qualify and quantify samples • Several hours to days Library construction • Prepare NGS library • Qualify and quantify library • ~4-8 hours Sequencing • Perform sequencing run • 8 hours to several days Data analysis • Primary, secondary data analysis • Several hours to days to… NGS Part 1: Introduction 22
- 23. Sample to Insight QIAGEN solution for NGS workflow Sample preparation Library construction Sequencing Data analysis • GeneRead® DNAseq NGS Panel System V2 • MagAttract® HMW DNA Kit • REPLI-g® Single Cell Kit • GeneRead™ rRNA Depletion Kit • GeneRead™ DNA QuantiMIZE • GeneRead™ DNA Library Prep • GeneRead Targeted Gene Panels • GeneRead™ Size Selection Kit • GeneRead™ Library Quant Kits Result validation • GeneRead DNAseq data analysis • Ingenuity Variant Analysis • RT2 Profiler PCR Arrays • Somatic Mutation PCR Arrays • PyroMark Pyrosequencing • CNA/CNV PCR Arrays • EpiTect ChIP PCR Arrays NGS Part 1: Introduction 23
- 24. Sample to Insight Agenda Next Generation Sequencing • Background • Technologies • Applications • Workflow Targeted Enrichment • Methodology • Data analysis • ??? NGS Part 1: Introduction 24 1 2
- 25. Sample to Insight The problem with patient samples • Low purity o Cancerous cells may be a minor fraction of total sample • Heterogeneity o Multiple sub-clones of cancer may be present in one tumor sample • Deep sequencing o 1000X coverage is required to get >90% sensitivity to detect ~5% mutation frequency Whole genome / exome sequencing is expensive and may not yield sufficient coverage NGS Part 1: Introduction 25
- 26. Sample to Insight What is targeted sequencing? • Sequencing a region or subset of the genome or transcriptome Why targeted sequencing? • Not all regions of the genome or transcripts are of interest or relevant to a specific study o Exome Sequencing: sequencing most of the coding regions of the genome (exome). The protein-coding region constitutes less than 2% of the entire genome o Focused panel/hot spot sequencing: focused on the genes or regions of interest e.g. Clinical relevance – tumor supressor genes, inherited mutations What are the advantages of targeted panel sequencing? • More coverage per sample, more sensitive mutation detection o 1 gene copy ~ 3 pg, 3000 copies in 10 ng o Heterogeneous sample 1% tumor cell = 30 copies in 10ng o Every base not covered equally in typical NGS experiment o Typically read 1000 reads per locus for somatic mutation o More samples per run, lower cost per sample Targeted sequencing NGS Part 1: Introduction 26
- 27. Sample to Insight Target enrichment: Methodology Hybridization capture • High input requirement (1 ug) • Long processing time (2-3 days) • Heterogeneous, lower specificity • Cover large regions (exome) NGS Part 1: Introduction 27 Data analysis Sequencing Hybridization capture (24-72 hrs) Library construction Sample preparation (DNA isolation)
- 28. Sample to Insight Target enrichment: Methodology Multiplex PCR • Small DNA input (< 100 ng) • No processing prior to enrichment • Short library prep time (<8 hrs) • Relatively small target region (KB - MB region) NGS Part 1: Introduction 28 Data analysis Sequencing Hybridization capture (24-72 hrs) Library construction Sample preparation (DNA isolation)
- 29. Sample to Insight QIAGEN GeneRead DNAseq Panel System V2 FOCUS ON YOUR RELEVANT GENES • Focused: o Biologically relevant content selection enables deep sequencing on relevant genes and identification of rare mutations • Flexible: o Mix and match any gene of interest or o Fully customizable panels available • NGS platform agnostic: o Functionally validated for Ion Torrent, MiSeq/HiSeq • Integrated controls: o Enabling quality control of prepared library before sequencing • Free, complete and easy of use data analysis tool NGS Part 1: Introduction 29
- 30. Sample to Insight GeneRead DNAseq V2 panel specifications Application Panel name # genes Target region (bases) Coverage (%) Specificity (%) Uniformity (%) Solid tumors Tumor Actionable Mutations 8 7,104 100.0 98.2 91 Clinically Relevant Tumor 24 39,603 98.1 95.3 90 Hematologic malignancies Myeloid Neoplasms 50 236,319 98.1 97.4 94 Disease-specific Breast Cancer 44 268,621 98.2 96.8 91 Colorectal Cancer 38 182,851 98.7 98.3 95 Liver Cancer 33 191,170 99.0 96.4 96 Lung Cancer 45 332,999 97.5 98.1 90 Ovarian Cancer 32 189,058 98.9 96.6 96 Prostate Cancer 32 167,195 98.4 97.3 94 Gastric Cancer 29 222,333 98.1 98.5 93 Cardiomyopathy 58 249,727 96.3 96.7 87 Comprehensive Carrier Testing 157 664,735 97.5 97.9 91 Cancer Predisposition 143 620,318 98.3 96.8 93 Comprehensive Cancer 160 744,835 98.0 97.7 92 Panel optimization results in outstanding experimental performance metrics NGS Part 1: Introduction 30
- 31. Sample to Insight GeneRead BRCA1 and BRCA2 custom panel Design and specifications Overlapping amplicon design 100% coverage Experimentally-verified 100% coverage Regions targeted Coding regions + 20 bp intron-exon junctions # of bases targeted 21,472 Average amplicon length 150 bp Total input DNA 40 ng Number of amplicons 250 Specificity 99% Uniformity (0.2x mean) 97% % of bases callable 100% NGS Part 1: Introduction 31
- 32. Sample to Insight GeneRead DNAseq Custom Panel Builder Build customized NGS panels in three simple steps List targets Customize View & Order NGS Part 1: Introduction 32
- 33. Sample to Insight GeneRead DNAseq Custom Panel Builder Quick start guide Enter targets Choose specs View design & Order Gene names Amplicon size: 150/225 Flanking bases: 10 (default) Chr Start Stop Enter name NGS Part 1: Introduction 33
- 34. Sample to Insight NGS data analysis • Base calling o From raw data to DNA sequences: generate sequencing reads • Mapping reads to a reference o Align the reads to reference sequence, e.g. GRCh37 o Similar to a BLAST search: compares millions of reads against a reference database • Variants identification o Identify the differences between sample DNA and reference DNA • Variant prioritization/filtering/validation/interpretation • Downstream validation o Typically PCR based, e.g. Somatic Mutation PCR analysis, methylation analysis, allele specific amplification. NGS Part 1: Introduction 34
- 35. Sample to Insight NGS data analysis: Alignment GRCh37 reference genome Sequencing reads A C C NGS Part 1: Introduction 35
- 36. Sample to Insight NGS data analysis: Sequencing depth • Coverage depth (or depth of coverage): how many times each base has been sequenced 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 0.001 – 0.01%) • Increasing coverage depth is also helpful to identify low frequent mutation in heterogenous samples such as identifying a somatic mutation in a heterogeneous cancer sample GRCh37 reference genome NGS reads coverage depth = 4 coverage depth = 2coverage depth = 3 NGS Part 1: Introduction 36
- 37. Sample to Insight 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 ROI 1 ROI 2 GRCh37 reference NGS reads NGS Part 1: Introduction 37
- 38. Sample to Insight NGS data analysis: Specificity 38 • Specificity: the percentage of sequences that map to the intended targets region of interest number of on-target reads / total number of reads ROI 1 ROI 2 Off-target reads On-target reads On-target reads GRCh37 reference NGS reads
- 39. Sample to Insight NGS data analysis: Uniformity • Coverage uniformity: measure the evenness of the coverage depth across target region o Calculate coverage depth of each position o Calculate the median coverage depth o Set the lower boundary of the coverage depth relative to median depth o (eg. 0.2 X median coverage depth in PCR panels) o Calculate the percentage of the target region covered to the depth of or deeper than the lower boundary GRCh37 reference NGS reads NGS Part 1: Introduction 39
- 40. Sample to Insight NGS data analysis: Uniformity • Coverage uniformity: measure the evenness of the coverage depth across target region o Calculate coverage depth of each position o Calculate the median coverage depth o Set the lower boundary of the coverage depth relative to median depth – (eg. 0.2 X median coverage depth in PCR panels) o Calculate the percentage of the target region covered to the depth of or deeper than the lower boundary coverage depth = 10 coverage depth = 2coverage depth = 3 GRCh37 reference NGS reads NGS Part 1: Introduction 40
- 41. Sample to Insight GeneRead data analysis solution • Data analysis for the non-bioinformaticians among us Input • FASTQ file • Panel ID • Job type o Single o Matched Tumor/Normal • Analysis mode o Somatic o Germline Output • Bam (sequence) and VCF (variant) files o Sequencing metrics o Variants detected, confidence o Copy number alterations • QIAGEN Advanced Bioinformatics (webinar IV) Comprehensive and easy-to-use data analysis NGS Part 1: Introduction 41
- 42. Sample to Insight NGS data analysis metrics Run Summary • Specificity • Coverage • Uniformity • Numbers of SNPs and indels Summary By Gene • Specificity • Coverage • Uniformity • # of SNPs and indels NGS Part 1: Introduction 42
- 43. Sample to Insight Features of Variant Report (QIAGEN) SNP detection Indel detection snpeff.sourceforge.net/ NGS Part 1: Introduction 43
- 44. Sample to Insight Confirm variants of your NGS runs Greek Symbol = Gene # = Mutation • For Normalization • Assays detect non- variable region (not ARMS- based design) qBiomarker Somatic Mutation ARMS-PCR Assays and Arrays NGS Part 1: Introduction 44
- 45. Sample to Insight GeneRead DNA NGS solutions from Sample to Insight NGS Part 1: Introduction 45
- 46. Sample to Insight Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: [email protected] [email protected] Questions? Thank you for attending NGS Part 1: Introduction 46
Editor's Notes
- #26: High level of experimental reproducibility. A REPLI-g WTA Single Cell reactions were performed on 3 (3 replicates), , using mRNA (poly A+) enrichment protocol to reduce rRNA amplification. . WTA Amplified cDNA was treated as decribed in figure xy and sequenced on a MiSeq Instrument (Illumina), RNA biotypes were mapped to single-transcript RNA using Bowtie2, and reads per kilobase and million mapped reads (RPKM) were calculated. Results demonstrate comparable average RPKM values of the 3-cell samples vs transcripts derived from WTA samples (10–50 cells). B REPLI-g WTA Single Cell reactions were performed on individual human cells including rRNA amplification. Real-time PCR of various transcripts (18S rRNA, 28S rRNA, ddx5, beta-actin, HPRT, GAPDH, PPIA, c-myc, RPS27a, BANF-1, abl-1) was done using QuantiFast SYBR Green PCR reagents and 1 ng of WTA-cDNA. Normalized CT values from two individual WTA reactions on single cells and the high R2 value > 0,97 demonstrate a high level of concordance in RNA amplification between experiments.