overview of ngs sequencing: methods and technologies

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


NGS

•Next Generation Sequencing

• “First generation sequencing”

→ Sanger sequencing


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



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore

454/Roche SoliD Complete Genomics Illumina Life Technologies

Pacific BioSciences Oxford Nanopore

•Second generation

•Third generation


NGS workflow

Library


NGS workflow

• Library preparation

•Sequencing

o clustering

o sequencing

•Data-analysis


NGS workflow


•Sequencing

o clustering

o sequencing

•Data-analysis


NGS Library Prep

10

TruSeq DNA Sample Prep Workflow

DNA RNA

Gel size selection, if needed


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


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


NGS Library Prep

10


DNA RNA



NGS Library Prep

• Indexing


NGS Library Prep

10


DNA RNA



NGS Library prep

•Fragmentation

o acoustic

covaris

o enzymatic


NGS Library prep

•Fragmentation

o quality check: BioAnalyser / FragmentAnalyser


NGS Library prep

•Standard A-tailing & adaptor-ligation (DNA/RNA)

10


DNA RNA



NGS Library Prep

• Illumina Nextera tagmentation

Introduction to NGS slide 35

© Céline Helsmoortel, Illumina, PacBio, Life Technologies, Oxford NanoPore

NGS workflow


•Sequencing

o clustering

o sequencing

•Data-analysis


NGS workflow

Library


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



11

NOTE:Single molecules bind to flow cell in a random pattern

Hybridize Fragment & Extend



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



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



13

Bridge Amplification

Single-stranded molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer

Hybridized primer is extended by polymerases



14


Double-stranded bridge is formed



15

Denature Double-stranded Bridge

Double-stranded bridge is denatured

Result:Two copies of covalently bound single-stranded templates



16


Single-stranded molecules flip over to hybridize to adjacent primers

Hybridized primer is extended by polymerase



17


Bridge amplification cycle repeated until multiple bridges are formed



18

Linearization

dsDNA bridges are denatured



19

Reverse Strand Cleavage

Reverse strands cleaved and washed away, leaving a cluster with forward strands only



20

Blocking

Free 3’ ends are blocked to prevent unwanted DNA priming



21

Read 1 Primer Hybridization

Sequencing primer

Sequencing primer is hybridized to adapter sequence



NGS workflow


•Sequencing

o clustering

o sequencing

•Data-analysis


NGS Library Prep

10


DNA RNA



NGS Illumina Sequencing

•Sequencing By Synthesis (SBS)



• 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



• 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



• 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



•Different SBS dyes


Overview of Illumina sequencers

MiSeq NextSeq HiSeq2500 HiSeq4000 NovaSeq

11





11





12









12









11







•Paired-end 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



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




33

Double stranded

DNA




34

Original forward strand


Bridges are linearized and the original forward template is cleaved



35

Reverse strand

template

Blocked 3’-ends

Sequencing primer


Free 3’ ends of the reverse template and lawn primers are blocked to prevent unwanted DNA priming

Sequencing primer is hybridized to adapter sequence



•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



•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:



•Quality Control



25

100 Microns

Clusters



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



•Quality Control


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



•Cluster generation HiSeq4000 & NovaSeq

o patterned flowcell- empty well-monoclonal well- polyclonal well

% occupied

% clusters passing filter



•Clusterdensity – patterned flow cells


PCR duplicates


Overview of Illumina sequencers

MiSeq NextSeq HiSeq2500 HiSeq4000 NovaSeq

11





11





12









12









11







•Quality Control



•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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•PhiX



NGS workflow


•Sequencing

o clustering

o sequencing

•Data-analysis


NGS workflow

Library


Data-analysis

raw BCL file fastQ file BAM file VCF file

demultiplexing mapping variant calling + annotation(indexes) (ex BWA) (ex GATK) (ex ANNOVAR)


Data-analysis




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


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 !


Data-analysis




Data-analysis

•BAM file (binary alignment/map)

ovisualisation in IGV

coverage


Data-analysis




Data-analysis

•VCF file (variant call format)

CS1 calls


NGS workflow

Genomics CoreCapita selecta Klinische biologie 15/10/2019Céline Helsmoortel

Thank you for your attention!

Questions?


NGS technologies



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore




•Third generation


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



Pacific BioSciences

PacBio® RS II Trace

SMRT® CellsZero-Mode Waveguides Phospholinked

Nucleotides

PacBio® Sequel


Pacific BioSciences


Pacific BioSciences

•Very long reads

•Accurate Hifi “shorter” reads


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

http://gc.uzleuven.be/


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)




PacBio RSII 40.000 – 70.000 reads

PacBio Sequel 350.000 - 600.000 reads


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


The Genomics Core

[email protected]


NGS technologies



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore




•Third generation


Life Technologies


Life Technologies66/128

Different platforms

Ion TorrentNext Generation Sequencing : Amplified Single Molecule Sequencing

4 nucleotides flow sequentially

No camera, just a pH sensor


Life Technologies

•Measurement of △pH @ base incorporation


NGS technologies



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore



First generation

(since 2005 …)

Second generation

Sanger sequencing


Roche/454 ABI SOLiD

Illumina


Pacific Biosciences

Third generation

Nanopore




•Third generation


Oxford Nanopore

overview of ngs sequencing: methods and technologies

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