The continuous evolution of NGS technology has led to an enormous diversification in NGS applications and dramatically decreased the costs to sequence a complete human genome.
In this presentation, we will discuss the following major topics:
• Basic overview of NGS sequencing technologies
• Next-generation sequencing workflow
• Spectrum of NGS applications
• QIAGEN universal NGS solutions
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Introduction to Next-Generation Sequencing (NGS) Technology
1. Sample to Insight
Introduction to Next-Generation Sequencing (NGS)
Wolfgang Krebs, R&D Scientist, QIAGEN
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Overview of NGS technologies and innovative NGS library
prep methods
Part 1: Introduction to next-generation sequencing (NGS)
technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Welcome to a 3-part series: NGS technology and applications
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3. Sample to Insight
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.
Legal disclaimer
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Illumina® with HiSeq® X in 2014… BUT data interpretation not included
Veritas Genetics in 2016 $1000 genome
Almost the $1000 genome….
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Data analysis
&
interpretation
Sequencing
Sample
extraction
Library
preparation
Genomic DNA
or RNA
Library preparation
Bring the DNA/RNA into a
format that is usable to the
sequencer
Sequencing on Illumina
or Ion Torrent™ platforms
Data analysis
The universal NGS workflow for Illumina/Ion Torrent
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1. Create DNA fragments
2. Add platform-specific adapter sequences to every fragment
3. Amplify library molecules (optional)
Library
preparation
Data analysis
&
interpretation
Sequencing
Sample
extraction
What NGS library prep accomplishes
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300 bp sample DNA insert
Adapter
ligation
point
Flowcell
What the adapters do #1: Bind library to a flowcell or bead
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Adapter
ligation
point
A C TG
A C TG
A C TG
A C TG
A C TG
Seq
primer 1
Seq primer
binding site
Bridge amplification
to form a cluster
Bind primer and sequence
What the adapters do #2: Add seq primer binding sites
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Barcode 1
Each 96-plex adapter includes two 8-base barcodes
8 barcode 1’s x 12 barcode 2’s = 96 combinations
Pool
barcoded
libraries
Single
multiplexed
NGS run
Demultiplexing
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
Separate FASTQ data files for each sample
Multiplexed sequencing: Multiple samples sharing one sequencing reaction to reduce
per-sample costs and control the amount of data generated per sample.
Adapter
ligation
point
What the adapters do #3: Add barcodes for multiplexing
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Adapter – A short double-stranded DNA fragment that is ligated to the sample DNA
fragments prior to sequencing.
Barcode – Unique molecular identifiers included in adapter sequences
.
Library complexity – A measure of the proportion of unique molecules within a library.
More complexity is better, and a high-complexity library is generally free of PCR
duplicates, adapter-dimers and other artifacts.
Multiplexing – Combining several different samples in one sequencing run.
Demultiplexing – The act of separating reads from an NGS run into separate piles
based on their index or barcode reads.
Key NGS vocabulary
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1st generation sequencing
• Sequence many identical molecules
• Sequencing in large gels or capillary
tubing limits scale
What came before next-generation sequencing?
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How is next-generation sequencing different?
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1st generation sequencing
• Sequence many identical
molecules
• Sequencing in large gels
or capillary tubing limits
scale
2nd generation sequencing
• Sequence millions of
clonally amplified molecules
per run
• Using a reversible, stepwise
sequencing chemistry
• Immobilized on a surface
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What comes after NGS?
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3rd generation sequencing
(NGS)
• Single molecule sequencing
in real-time
• Generation of very long
reads (>>5 kb)
• Faster than 2nd gen (hours
instead of days)
• Still high on error rates
1st generation sequencing
• Sequence many identical
molecules
• Sequencing in large gels
or capillary tubing limits
scale
2nd generation sequencing
• Sequence millions of
clonally amplified molecules
per run
• Using a reversible, stepwise
sequencing chemistry
• Immobilized on a surface
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Example: Illumina platforms
1. Cluster amplification 2. Sequencing
What happens on the sequencer?
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Source: Cram genomics http://www.cram.com/flashcards/genomics-6416574
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• DNA fragments are flanked with adaptors (library)
• A solid surface is coated with primers complementary to the two adaptor sequences
• Isothermal 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
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Bridge amplification (isothermal amplification)
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• Run time: 1–10 days
• Produces: 2–1000 Gb of sequence
• Read length: 2 x 50 bp – 2 x 250 bp
(paired-end)
• Cost: $0.05–$0.40/Mb
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Illumina HiSeq/MiSeq®
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Image source: Nature Reviews 11. http://www.nature.com/nrg/journal/v11/n1/images/nrg2626-f2.jpg
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Single-end reading (SE):
• Sequencer reads a fragment from only one primer binding site
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
• Required for high-order multiplexing of samples (indexes on both sides)
Single-end
reading
2nd strand
synthesis
Paired-end
reading
23
Illumina single-end vs. paired-end
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• 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 deposited into the wells of sequencing chips – one well,
one bead
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Emulsion PCR: Ion Torrent
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Image source: Shendure, J. and Hanlee, J. (2008) Next-generation DNA sequencing. Nature Biotechnology 26, 1135–45.
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• Run time: 3 h; no termination or deprotection steps
• Read length: 100–300 bp
• Throughput determined by chip size (pH meter array): 10Mb – 5 Gb
• Cost: $1–$20/Mb
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Ion Torrent PGM/Proton
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Image sources: Rothberg, J.M., et al. (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–52.
Tinning, M. (2012) Next gen sequencing platforms and applications. Australian Genome Research Facility slideshare. http://www.slideshare.net/AGRF_Ltd/ngs-
technologies-platforms-and-applications?qid=d1e6ef45-c2de-41e5-8fa8-c7872ced32d3&v=&b=&from_search=1
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NGS – Next-generation sequencing.
Flow cell – Name of the Illumina sample chip. Ready-to-sequence libraries are
bound to the flow cell and bridge amplified prior to sequencing. Depending on the
type of sequencer and the number of samples, different flow cell sizes are
available.
FASTQ file – default file format for sequencer output.
Alignment – the act of “matching” reads to a reference genome.
Key NGS vocabulary
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Application areas
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Source: Rizzo, J.M. and Buck, M.J. (2012) Key principles and clinical applications of “next-generation” DNA sequencing. Cancer Prev Res (Phila) 5, 887–900.
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Large fraction of genome = greater costs, but greater ability to compare samples or patients across studies
Small targeted panels = easier and less expensive to complete the project, but limited ability to follow up on new
hypotheses or discover new associations
Whole
Genome
Exome
Sequencing
DNA Targeted
Panels
RNA-Seq
RNA targeted panels
• Gene expression
• Gene fusions
• Splice variants (known targets)
Four key NGS applications
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Image source: Simon, R. and Roychowdhury, S. (2013) Implementing personalized cancer genomics in clinical trials. Nature 12, 358–69.
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AnalyteSample Type
Blood
Tissue
Metagenomics
Blood cells
Circulating cell-free
nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
FFPE
Fresh frozen
Single cells
Stool, soil, water, etc.
NGS applications – Details
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AnalyteSample Type
Blood
Tissue
Metagenomics
Blood cells
Circulating cell-free
Nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
DNA
RNA
Epigenetics
FFPE
Fresh Frozen
Single Cells
Stool, soil, water, etc.
NGS applications – Details
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AnalyteSample Type
Blood cells
Circulating cell-free
nucleic acids
Circulating tumor cells
(CTCs)
Exosomes
DNA
RNA
Epigenetics
FFPE
Fresh frozen
Single cells
Stool, soil, water, etc.
Sequencing
Targeted
Non-targeted
NGS applications – Details
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Targeted Sequencing
PCR-based target enrichment
(amplicon sequencing)
Hybrid capture target enrichment
(whole exome, targeted regions)
Lib prep: End repair, A-tailing,
adapter ligation
PCR primers, target
enrichment
Adapter
llgation
point
T
Shearing of gDNA
(enzymatic, mechanical)
Lib prep: End repair, A-tailing,
adapter ligation
Hybrid capture with target-specific
probes;
enrich for
target regions
Extracted DNA
Hybrid capture vs. amplicon sequencing
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Whole
genome
Exome
sequencing
RNA-Seq
DNA
targeted
panels
All bases,
low depth
~5–100 M reads
(variable)
RNA
targeted
panels
90 Gb
(30x human)
~25 M reads
(100x)
~2 M reads*
(1000x)
Few bases,
high depth
ChIP-
Seq
~5 M reads
(variable)
~2 M reads
(1000x)
RNA
DNA
Gb = # reads x read length
* Depends on gene number and sequencing depth
Typical coverage or sequencing depth for key NGS applications
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Capacity of different sequencers is defined by number of reads generated per run
(sequencer throughput)
Illumina
Thermo
Fisher
MiniSeq™
15–50 M
7.5 Gb
MiSeq
25–50 M
15 Gb
NextSeq™ 500
400 M
120 Gb
HiSeq 2500
4000 M
1000 Gb
HiSeq X
6000 M
1800 Gb
PGM
5 M
2 Gb
S5/S5XL
80 M
15 Gb
Low
throughput
High
throughput
Sequencers have different capacities (throughput)
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RNA targeted panels
DNA targeted panels
mRNA-Seq
ChIP-seq Exome sequencing
Whole genome(Small WGS)
The sequencer is an indicator for the NGS application
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Illumina
MiniSeq™ MiSeq NextSeq™ 500 HiSeq 2500 HiSeq X
Thermo
Fisher
PGM S5/S5XL
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Coverage – The proportion of a reference genome that has reads aligned to it.
Sequencing depths – Refers to the number of reads that cover a given position in a
reference.
Whole genome sequencing – Sequencing as much of the genome as possible to either
assemble a draft genome for new organisms or to identify sequence variants, chromosomal
rearrangements or other structural variants for organisms with a reference.
Exome sequencing = hybrid capture sequencing – Sequence only a subset of the genome
containing features of interest, which are selected out of a whole genome library using hybrid
capture.
Targeted panel sequencing = amplicon panel sequencing = targeted resequencing –
Regions of interest are amplified from gDNA or cDNA via PCR, and the amplicons are
sequenced. Offers greater sequencing depth than WGS, but focus on a small set of genomic
regions.
Key NGS vocabulary
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The QIAseq portfolio covers a broad range of NGS applications
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Explore QIAseq NGS solutions: https://www.qiagen.com/products/ngs/ngs-life-sciences/
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QIAseq NGS solutions
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Focus products for whole genome or exome sequencing
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NGS
Library
preparation
NGS
library
preparation
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Overview of NGS technologies and innovative NGS library
prep methods
Part 1: Introduction to next-generation sequencing (NGS)
technology
Part 2: Innovative NGS library construction technology
Part 3: Advanced NGS library prep for challenging samples
Upcoming webinars
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Questions?
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QIAGEN.com
Contact QIAGEN technical service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 EU
Email:
techservice-na@QIAGEN.com
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QIASeq.NGS@QIAGEN.com
QIAwebinars@QIAGEN.com
Thank you for attending!
Intro to NGS, 11.30.2016
Notas do Editor
In the past (10–15 years ago), sequencing may have been a more rarely used academic method – or a method only reserved for rich people desiring a personalized diagnostic. In 2016, the technique has become a standardized method to detect genetic diseases or cancer and has revolutionized the biomedical research and personalized healthcare. McKinsey listed next-generation sequencing (NGS) as one of the 12 technologies that will transform our lives.
HiSeq 2000 0.2 10E+9 Kb
HiSeq X System 0.9-1.8 10E+9 Kb almost 10 times more than HiSeq2000
Drastic decrease in sequencing costs per genome.
To illustrate the nature of the reductions in DNA sequencing costs, each graph also shows hypothetical data reflecting Moore's Law, which describes a long-term trend in the computer hardware industry that involves the doubling of “computer power” every two years (See: Moore's Law [wikipedia.org]). Technology improvements that keep up with Moore's Law are widely regarded to be doing exceedingly well, making it useful for comparison.
Moore’s law square.
A very high proportion of current NGS sequencers use platforms from Illumina and Ion Torrent.
Thus, I will describe a general NGS workflow applicable for these two sequencer types – which require a library preparation step, followed by performance of the sequencing reaction, itself, with sequencing-by-synthesis technology.
Due to less efficient library construction – or due to the multiple PCR cycles required for library amplification – complexity can potentially decrease. A higher complexity means more unique molecules within an NGS library, which is always preferred to a lower complexity.
[Mention our CLC Analysis software]
Makes our Sample to Insight mission possible.
Used either chemical or enzymatic methods to generate a nested set of DNA fragments.
Used electrophoretic methods to separate the fragments.
Required lots of DNA (100s of ng to 1 ug), so it typically involved cloning and/or PCR.
Limited scale and throughput.
Sequencers on the picture:
Life Technologies/Applied Biosystems; now part of Thermo Fisher Scientific, SOLID 5500
Illumina MiSeq
Ion Torrent PGM, also now part of Thermo Fisher Scientific
Roche / 454 Pyrosequencer
QIAGEN GeneReader
--Pacific Biosciences RS II System / Sequel System (pictures 1+2) with the SMRT (single molecule real-time) sequencing technology
Zero-Mode Waveguides (ZMWs) allow light to illuminate only the bottom of a well in which a DNA polymerase/template complex is immobilized. Phospholinked nucleotides allow observation of the immobilized complex as the DNA polymerase produces a completely natural DNA strand
Allows simultaneous epigenetic characterization (modified vs. unmodified bases)
--Oxford Nanopore (pictures 3+4, “Pocket-sized” minION and “Desktop” PromethION [not available, only in early access program])
The Nanopore system uses a technique that passes intact DNA polymers through a protein nanopore, sequencing in real time as the DNA translocates the pore. DNA is sequenced by measuring the change in current across the membrane when the DNA passes through the pore; different base pairs have different conductance, and thus different signals.
FFPE = Formalin-fixed paraffin-embedded
Especially for targeted panels, the read counts and sequencing depth depends not only on the type of the panel with a lower or higher number of target regions but also on the type of analysis. A very sensitive variant calling analysis with 1% detection threshold needs a higher sequencing converage in comparison to a 5% threshold analysis.