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 â 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
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 âbridgeâ 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 â 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 (âmappedâ)
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 â no termination and deprotection steps
⢠Read length 100â300 bp; homopolymer can be an issue
⢠Throughput determined by chip size (pH meter array): 10Mb â 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
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 toâŚ
NGS Part 1: Introduction 22
23. Sample to Insight
QIAGEN solution for NGS workflow
Sample
preparation
Library
construction
Sequencing
Data analysis
⢠GeneReadŽ DNAseq NGS Panel System V2
⢠MagAttractŽ HMW DNA Kit
⢠REPLI-gŽ 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
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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 â 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
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GeneRead DNAseq Custom Panel Builder
Build customized NGS panels in three simple steps
List targets
Customize
View & Order
NGS Part 1: Introduction 32
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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
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NGS data analysis: Alignment
GRCh37 reference genome
Sequencing reads
A
C
C
NGS Part 1: Introduction 35
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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 â 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
â (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)
ďŽ SNP detection
ďŽ Indel detection
snpeff.sourceforge.net/
NGS Part 1: Introduction 43
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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
46. Sample to Insight
Thank you for attending todayâs webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email:
techservice-na@qiagen.com
BRCsupport@QIAGEN.com
Questions?
Thank you for attending
NGS Part 1: Introduction 46
Hinweis der Redaktion
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 (10â50 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.
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