overview of ngs sequencing: methods and technologies
TRANSCRIPT
Genomics CoreCapita selecta Klinische biologie 15/10/2019Céline Helsmoortel
Overview of NGS sequencing: methods and technologies
Introduction to NGS slide 3© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS
•Next Generation Sequencing
Introduction to NGS slide 4© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS
•Next Generation Sequencing
• “First generation sequencing”
→ Sanger sequencing
Introduction to NGS slide 5© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS technologies
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
454/Roche SoliD Complete Genomics Illumina Life Technologies
Pacific BioSciences Oxford Nanopore
•Second generation
•Third generation
Introduction to NGS slide 7© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
Library
Introduction to NGS slide 8© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
• Library preparation
•Sequencing
o clustering
o sequencing
•Data-analysis
Introduction to NGS slide 9© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
• Library preparation
•Sequencing
o clustering
o sequencing
•Data-analysis
Introduction to NGS slide 10© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
10
TruSeq DNA Sample Prep Workflow
DNA RNA
Gel size selection, if needed
Introduction to NGS slide 11© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Illumina flowcells
9
Cluster Generation
Library pool loaded into reagent cartridge flows through all 4 lanes of the flow cell
Hybridization and cluster generation are automated on the NextSeq system
Approximately 5,000 molecules are included in a cluster
Single DNA Library
Amplified Clonal
Cluster
12
Flow Cell Design
Dual-surface enabled• 8 lane format• 25mm wide x 75mm long• 1.7mm-wide lanes• Only compatible with cBot
MiSeq
NextSeq
Rapid High OutputHiSeq
NovaSeq
SP
Introduction to NGS slide 12© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
8
What is a Flow Cell?
A flow cell is a thick glass slide with channels or lanes
Each lane is randomly coated with a lawn of oligos that are complementary to library adapters
Cluster generation occurs on a flow cell
P5 P7
Introduction to NGS slide 13© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
10
TruSeq DNA Sample Prep Workflow
DNA RNA
Gel size selection, if needed
Introduction to NGS slide 14© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
• Indexing
Introduction to NGS slide 15© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
10
TruSeq DNA Sample Prep Workflow
DNA RNA
Gel size selection, if needed
Introduction to NGS slide 18© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library prep
•Fragmentation
o acoustic
covaris
o enzymatic
Introduction to NGS slide 19© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library prep
•Fragmentation
o quality check: BioAnalyser / FragmentAnalyser
Introduction to NGS slide 20© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library prep
•Standard A-tailing & adaptor-ligation (DNA/RNA)
10
TruSeq DNA Sample Prep Workflow
DNA RNA
Gel size selection, if needed
Introduction to NGS slide 21© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
• Illumina Nextera tagmentation
Introduction to NGS slide 35
© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
• Library preparation
•Sequencing
o clustering
o sequencing
•Data-analysis
Introduction to NGS slide 36© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
Library
Introduction to NGS slide 37© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
Introduction to NGS slide 38© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
12
Flow Cell Design
Dual-surface enabled• 8 lane format• 25mm wide x 75mm long• 1.7mm-wide lanes• Only compatible with cBot
Introduction to NGS slide 39© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
11
NOTE:Single molecules bind to flow cell in a random pattern
Hybridize Fragment & Extend
Introduction to NGS slide 40© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
10
Hybridize Fragment & Extend
Adapter sequence
3’ extension
Surface of flow cell coated with a lawn of oligo pairs
Single DNA libraries are hybridized to primer lawn
Bound libraries then extended by polymerases
Introduction to NGS slide 41© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
12
Newly synthesized
strandOriginal template
Denature Double-stranded DNA
discard
Double-stranded molecule is denatured
Original template washed away
Newly synthesized strand is covalently attached to flow cell surface
Introduction to NGS slide 42© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
13
Bridge Amplification
Single-stranded molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer
Hybridized primer is extended by polymerases
Introduction to NGS slide 43© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
14
Bridge Amplification
Double-stranded bridge is formed
Introduction to NGS slide 44© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
15
Denature Double-stranded Bridge
Double-stranded bridge is denatured
Result:Two copies of covalently bound single-stranded templates
Introduction to NGS slide 45© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
16
Bridge Amplification
Single-stranded molecules flip over to hybridize to adjacent primers
Hybridized primer is extended by polymerase
Introduction to NGS slide 46© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
17
Bridge Amplification
Bridge amplification cycle repeated until multiple bridges are formed
Introduction to NGS slide 47© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
18
Linearization
dsDNA bridges are denatured
Introduction to NGS slide 48© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
19
Reverse Strand Cleavage
Reverse strands cleaved and washed away, leaving a cluster with forward strands only
Introduction to NGS slide 49© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
20
Blocking
Free 3’ ends are blocked to prevent unwanted DNA priming
Introduction to NGS slide 50© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
21
Read 1 Primer Hybridization
Sequencing primer
Sequencing primer is hybridized to adapter sequence
Introduction to NGS slide 51
© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
• Library preparation
•Sequencing
o clustering
o sequencing
•Data-analysis
Introduction to NGS slide 52© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Library Prep
10
TruSeq DNA Sample Prep Workflow
DNA RNA
Gel size selection, if needed
Introduction to NGS slide 53© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Sequencing By Synthesis (SBS)
Introduction to NGS slide 54© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
• Imaging HiSeq/MiSeq
15
HiSeq System Imaging
Operates in epi-illumination mode
• Fluorescence and emission from the same side of the sample
Continuous Scanning• Cameras operate in time delay
integration (TDI)• Fluorescence image continually
read
Dual Surface Scanning• First: Top of flow cell, all lanes
• Second: Bottom of flow cell, all lanes
3 swaths/lane + 2 surfaces/lane = 6 scanning events/lane
Introduction to NGS slide 55© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
• Imaging MiSeq, HiSeq2500, HiSeq4000
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
Introduction to NGS slide 56© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
• Imaging NextSeq, NovaSeq
12
Two channel SBS uses 2 images
Builds template over 5 cycles
Clusters appearing in green only are T
Clusters appearing in red only are C
Clusters appearing in both images are A
Clusters not present in either green nor red are G
Cluster intensities are plotted and bases are called accordingly
Two Channel SBS – NextSeq Series
Introduction to NGS slide 57© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Different SBS dyes
Introduction to NGS slide 58© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Overview of Illumina sequencers
MiSeq NextSeq HiSeq2500 HiSeq4000 NovaSeq
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
12
Two channel SBS uses 2 images
Builds template over 5 cycles
Clusters appearing in green only are T
Clusters appearing in red only are C
Clusters appearing in both images are A
Clusters not present in either green nor red are G
Cluster intensities are plotted and bases are called accordingly
Two Channel SBS – NextSeq Series
12
Two channel SBS uses 2 images
Builds template over 5 cycles
Clusters appearing in green only are T
Clusters appearing in red only are C
Clusters appearing in both images are A
Clusters not present in either green nor red are G
Cluster intensities are plotted and bases are called accordingly
Two Channel SBS – NextSeq Series
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
Introduction to NGS slide 59© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Paired-end sequencing
Introduction to NGS slide 60© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
31
Sequenced strand
Blocked 3’-ends
Sequenced strand is stripped off
3’-ends of template strands and lawn primers are unblocked
Paired End Sequencing
Introduction to NGS slide 61© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
32
Bridge formation
3’ extension
Single-stranded template loops over to form a bridge by hybridizing with a lawn primer
3’-ends of lawn primer is extended
Paired End Sequencing
Introduction to NGS slide 62© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
33
Double stranded
DNA
Paired End Sequencing
Introduction to NGS slide 63© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
34
Original forward strand
Paired End Sequencing
Bridges are linearized and the original forward template is cleaved
Introduction to NGS slide 64© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
35
Reverse strand
template
Blocked 3’-ends
Sequencing primer
Paired End Sequencing
Free 3’ ends of the reverse template and lawn primers are blocked to prevent unwanted DNA priming
Sequencing primer is hybridized to adapter sequence
Introduction to NGS slide 66© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Dual index sequencing
40
Sequencing Paired End Libraries with Dual Index Read
1 2 3
Paired End Turnaround
4
Dual Index Sequencing Utilizes 4 Sequencing Reads
Introduction to NGS slide 68© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Dual index sequencing NextSeq & HiSeq4000 PE
15
Index read 3
i5 Index Seq Primer (SBS3rev/BP13)
i5 Index
Index Read 2 (i5)
i7 Index 1 2 3
Paired End Turnaround
4
Sequencing Paired End Libraries with Dual Index Read: NextSeq
• Dual-indexed single reads will need to go through paired end turn around • During run set up, do not enter cycles for read 2
Note on NextSeq Single Read Dual Index Sequencing:
Introduction to NGS slide 69© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Quality Control
Introduction to NGS slide 70© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Quality Control
Introduction to NGS slide 71© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Quality Control
Introduction to NGS slide 72© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
25
100 Microns
Clusters
Introduction to NGS slide 73© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
25
Maximize data quality and quantity
20pM 10pM 5pM 1pM
Optimized flow cell clustering determines data quality and overall data yield
Overclustering can result in:• Loss of data quality and data output• Loss of focus• Reduced base calls and Q30 scores• Complete run failure
Underclustering can result in:• Loss of time and money
•Clusterdensity – non patterned flow cells
Introduction to NGS slide 74© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Quality Control
Introduction to NGS slide 75© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Clusters Passing Filter
Introduction to NGS slide 76© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Clusters Passing Filter
•Cluster generation MiSeq, NextSeq, HiSeq2500
•Chastity filter
21 FOR RESEARCH USE ONLY
Process of Extracting Intensity Part 1
Cluster A
threshold
Cluster B
Values below threshold are not considered
Only a small area of the signal is considered to avoid cluster overlap
Introduction to NGS slide 77© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Clusters Passing Filter
•Cluster generation HiSeq4000 & NovaSeq
o patterned flowcell- empty well-monoclonal well- polyclonal well
% occupied
% clusters passing filter
Introduction to NGS slide 78© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Clustering
•Clusterdensity – patterned flow cells
Introduction to NGS slide 79© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
PCR duplicates
Introduction to NGS slide 80© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Overview of Illumina sequencers
MiSeq NextSeq HiSeq2500 HiSeq4000 NovaSeq
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
12
Two channel SBS uses 2 images
Builds template over 5 cycles
Clusters appearing in green only are T
Clusters appearing in red only are C
Clusters appearing in both images are A
Clusters not present in either green nor red are G
Cluster intensities are plotted and bases are called accordingly
Two Channel SBS – NextSeq Series
12
Two channel SBS uses 2 images
Builds template over 5 cycles
Clusters appearing in green only are T
Clusters appearing in red only are C
Clusters appearing in both images are A
Clusters not present in either green nor red are G
Cluster intensities are plotted and bases are called accordingly
Two Channel SBS – NextSeq Series
11
Four Channel SBS Chemistry: GA, HiSeq, MiSeq
Collects 4 images • During each cycle,
each cluster appears in only 1 of 4 images
Each of the 4 DNA bases emit an intensity of a unique wavelength
Introduction to NGS slide 81© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•Quality Control
Introduction to NGS slide 82© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•PhiX
o artificial phage
o small genome: quick sequencing & alignment
→ estimation of error rate
o diverse: ~45% GC and 55% AT
o well known genome
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Introduction to NGS slide 83© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS Illumina Sequencing
•PhiX
Introduction to NGS slide 84
© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
• Library preparation
•Sequencing
o clustering
o sequencing
•Data-analysis
Introduction to NGS slide 85© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
Library
Introduction to NGS slide 86© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
raw BCL file fastQ file BAM file VCF file
demultiplexing mapping variant calling + annotation(indexes) (ex BWA) (ex GATK) (ex ANNOVAR)
Introduction to NGS slide 87© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
raw BCL file fastQ file BAM file VCF file
demultiplexing mapping variant calling + annotation(indexes) (ex BWA) (ex GATK) (ex ANNOVAR)
Introduction to NGS slide 88© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
•FastQ fileIllumina FASTQ files
Divided into blocks of 4 lines
Machine ID Run ID Lane Tile
X pos Y pos
1. ID 2. Sequence 4. Quality
Introduction to NGS slide 89© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
•FastQ fileFormat of Illumina FASTQ files can greatly vary
Here quality scores are represented as ASCII characters (ASCII offset 33)
-> You have to add 33 to the quality Phred score
e.g. quality score = 38 ; 38 + 33 = 71 ; character 71 in ASCII code = G
Always check which ASCII version is used !
Introduction to NGS slide 90© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
raw BCL file fastQ file BAM file VCF file
demultiplexing mapping variant calling + annotation(indexes) (ex BWA) (ex GATK) (ex ANNOVAR)
Introduction to NGS slide 91© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
•BAM file (binary alignment/map)
ovisualisation in IGV
coverage
Introduction to NGS slide 92© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
raw BCL file fastQ file BAM file VCF file
demultiplexing mapping variant calling + annotation(indexes) (ex BWA) (ex GATK) (ex ANNOVAR)
Introduction to NGS slide 93© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Data-analysis
•VCF file (variant call format)
CS1 calls
Introduction to NGS slide 94© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS workflow
Introduction to NGS slide 95© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Genomics CoreCapita selecta Klinische biologie 15/10/2019Céline Helsmoortel
Thank you for your attention!
Questions?
Introduction to NGS slide 100© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS technologies
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
454/Roche SoliD Complete Genomics Illumina Life Technologies
Pacific BioSciences Oxford Nanopore
•Second generation
•Third generation
Introduction to NGS slide 101© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Observe single molecules in real time to provide high-throughput SMRT® Sequencing of DNA and base modifications simultaneously
Pacific BioSciences
• Generate finished genomes
• Discover a broad spectrum of base modifications
• Characterize complex variations
• Long read lengths
• High accuracy
• Ultra sensitivity
• Shortest run time
• Least GC bias
• No amplification bias
Introduction to NGS slide 102
© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Pacific BioSciences
PacBio® RS II Trace
SMRT® CellsZero-Mode Waveguides Phospholinked
Nucleotides
PacBio® Sequel
Introduction to NGS slide 103© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Pacific BioSciences
Introduction to NGS slide 104© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Introduction to NGS slide 105© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Pacific BioSciences
•Very long reads
•Accurate Hifi “shorter” reads
Introduction to NGS slide 106© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS @ Genomics Core
• Create a project on http://www.genomicscore.be/request
• Specifyo project description
o sample type (DNA / RNA / library / …)
o sample origino concentration, average fragment size (libraries @ 4nM)
o indexes
o sequencing requirements (PE100, SR50, …)o bioinformatics
o transfer of results
Introduction to NGS slide 107© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS @ Genomics Core
Instrument / kit Sequencing Capacity (# reads)
MiSeq v2 Nano 1 M
MiSeq v2 Micro 4 M
MiSeq v2 Standard 15 M
MiSeq v3 25 M
NextSeq Mid Output 130 M
NextSeq High Output 400 M
HiSeq2500 Rapid run v2 150 M / lane (2 lanes)
HiSeq2500 High Output run v2 250 M / lane (8 lanes)
HiSeq4000 320 M / lane (8 lanes)
NovaSeq SP 400 M / lane (2 lanes)
NovaSeq S1 800 M / lane (2 lanes)
NovaSeq S2 1650 M / lane (2 lanes)
NovaSeq S3 1650 M / lane (4 lanes)
NovaSeq S4 2500 M / lane (4 lanes)
PacBio RSII 40.000 – 70.000 reads
PacBio Sequel 350.000 - 600.000 reads
Introduction to NGS slide 108© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS @ Genomics Core
Centrum Menselijke Erfelijkheid Constitutionele Cytogenetica
GC Toepassingsdatum: Zie metadata MuzliDoc 1/1
AANVRAAGFORMULIER LIBRARIES GENOMICS CORE
LABO:________________________ LABO: GC
AANVRAAG:________________________ ONTVANGST
� In diepvries GC geplaatst
door:
op: ___ /___ / 201__
om ____ u ____ min
� GC code(s) aanvraag:
� Aangeleverde concentratie staal: ………………………nM
� Eindconcentratie staal op sequencer: ………………..pM
� Percentage PhiX: …………………………%
� Custom primers: JA / NEEN
- Naam en eindconc.: …………….………………..…, …………µM
- Enkel custom primers of spike-in bij illumina primers?
� Type kit:
v2 nano / v2 micro / v2 standaard / v3
� Aantal cycli
(read1 – index1 – optioneel index2 – read2):
…………….. - …………….. - …………….. - ……………..
� Opmerkingen:
� In diepvries GC ontvangen
door:
op: ___ /___ / 201__
om ____ u ____ min
� GC code op etiket staal:
� Identificatie staal OK: JA / NEEN
� GC code als ‘ontvangen’
geregistreerd in GC database:
JA / NEEN
� Opmerkingen:
GC-MT009-AN01 M04MT231-AN01
Centrum Menselijke Erfelijkheid Constitutionele Cytogenetica
GC Toepassingsdatum: Zie metadata MuzliDoc 1/1
AANVRAAGFORMULIER LIBRARIES GENOMICS CORE
LABO:________________________ LABO: GC
AANVRAAG:________________________ ONTVANGST
� In diepvries GC geplaatst
door:
op: ___ /___ / 201__
om ____ u ____ min
� GC code(s) aanvraag:
� Aangeleverde concentratie staal: ………………………nM
� Eindconcentratie staal op sequencer: ………………..pM
� Percentage PhiX: …………………………%
� Custom primers: JA / NEEN
- Naam en eindconc.: …………….………………..…, …………µM
- Enkel custom primers of spike-in bij illumina primers?
� Type kit:
v2 nano / v2 micro / v2 standaard / v3
� Aantal cycli
(read1 – index1 – optioneel index2 – read2):
…………….. - …………….. - …………….. - ……………..
� Opmerkingen:
� In diepvries GC ontvangen
door:
op: ___ /___ / 201__
om ____ u ____ min
� GC code op etiket staal:
� Identificatie staal OK: JA / NEEN
� GC code als ‘ontvangen’
geregistreerd in GC database:
JA / NEEN
� Opmerkingen:
GC-MT009-AN01 M04MT231-AN01
Introduction to NGS slide 109© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
The Genomics Core
Introduction to NGS slide 110© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS technologies
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
454/Roche SoliD Complete Genomics Illumina Life Technologies
Pacific BioSciences Oxford Nanopore
•Second generation
•Third generation
Introduction to NGS slide 111© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Life Technologies
Introduction to NGS slide 112© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Life Technologies
Introduction to NGS slide 113© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Life Technologies66/128
Different platforms
Ion TorrentNext Generation Sequencing : Amplified Single Molecule Sequencing
4 nucleotides flow sequentially
No camera, just a pH sensor
Introduction to NGS slide 114© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Life Technologies
•Measurement of △pH @ base incorporation
Introduction to NGS slide 115© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
NGS technologies
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
Chain-termination (1980s)
Dye-termination (1990s) automatic capillary (~800 bp)
First generation
(since 2005 …)
Second generation
Sanger sequencing
Traditional sequencing versus NGS equipment
Roche/454 ABI SOLiD
Illumina
CompleteGenomics Iron Torrent
Pacific Biosciences
Third generation
Nanopore
454/Roche SoliD Complete Genomics Illumina Life Technologies
Pacific BioSciences Oxford Nanopore
•Second generation
•Third generation
Introduction to NGS slide 116© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Oxford Nanopore
Introduction to NGS slide 117© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore
Oxford Nanopore