basic principles of whole genome sequencing

Basic Principles of Whole Genome

Sequencing

© ESCMID eLibrary by a

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Disclosure of speaker’s interests

(Potential) conflict of interest

none

Potentially relevant company relationships in

connection with event 1

none

Sponsorship or research funding2

Fee or other (financial) payment3

Shareholder4

Other relationship, i.e. …5

none

Disclosure slide for speaker at EUCIC Advanced module

for Infection Prevention and Control


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Why?

• Define differences

• Outbreak situation & transmission chains

• Endemic situation – Monitoring/Surveillance

• Diagnosis (Re-Infection?)

• Nomenclature

• Difference epidemic clones with special characteristics

• Virulence factors, antibiotic resistance pattern

• Requirements: Discriminatory, specific, reproducible

• Fingerprint

Bacterial Diversity and Typing


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How?

• Non-molecular methods

• Phage-Typing

• Phenotyping

• Serology

• Molecular methods

• DNA banding pattern (e.g. AFLP)

• Sequence-based analyses (e.g. MLST, spa-typing)

• PCR detection



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Sequence-based molecular typing

• Multi-locus sequence typing (MLST)

• Differences in 7 housekeeping genes

• Combination of alleles resulting in sequence types (ST) – phone number

• ST1 – 1-1-1-1-1-1-1

• ST22 – 7-6-1-5-8-8-6

• Sequence types with 5 or more shared alleles are combined in clonal complexes (CC) – area code

• CC22

• ST22 7-6-1-5-8-8-6

• ST2371 258-6-1-5-8-8-6

• ST337 215-6-184-5-8-8-6

• ST3924 411-6-1-5-8-8-6



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With epidemic clones circulating, typing is at its limit

• EMRSA-15, K. pneumoniae ST258, E. coli ST131

@torstenseemann



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CC5

CC1

CC8

CC22 – EMRSA-15

CC45

CC30 – EMRSA-16

MSSA Addenbrooke’s

Phylogeny – Staphylococcus sp.


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@torstenseemann


With epidemic clones circulating, typing is at its limit

• EMRSA-15, K. pneumoniae ST258, E. coli ST131


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Whole genome sequencing (WGS)

• Species

• Subtypes

• Antibiotic resistance

• Known genes & mutations, future predictions possible

• Virulence factors

• Phylogenetic relationships



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• 1. Generation – “old school”

Sanger Sequencing – 1977

ABI Prism 3700

Overview of Sequencing Generations


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Background: DNA

Double-stranded molecule

Nucleoside-Monophosphate + Base

Chain

(RNA only) (DNA only)


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Background: DNA

DNA double strand

Base pairing


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Chain termination Synthesis

Background: Sanger Method

RNA Ribonucleic acid

DNA Deoxyribonucleic acid

dDNA Dideoxyribonucleic acid

O B

O

O

O

O-

P

OH OH

O B

O

O

O

O-

P

OH H

O B

O

O

O

O-

P

H H

5’ 5’ 5’

3’ 3’ 3’

2’Hydroxy 2’Deoxy Di-deoxy


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Background: Sanger Method

https://lerninhalte.blogspot.com/2013/04/dna-sequenzierung-kettenabbruchmethode.html

Rea

din

g d

irec

tio

n

Sequence to analyse

Radioactively labelled primer

Electro-phoresis


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Human Genome Project (1990-2003)

Capillary sequencing instead of gel electrophoresis (ca. 1998)

1. Generation: ABI3700 - 1998

LiCor ABI Prism 3700


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ABI Prism 3700

• 2. Generation – ”Next-generation” sequencing

Illumina MiSeq/HiSeq

454 (✝), IonTorrent, Qiagen GeneReader

Massive, parallel Shot-gun sequencing



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DNA Fragmentation, PCR Amplification

Background: Shot-gun Sequencing

BAC: Amplification Bias (systematic error)

Shot-gun: Amplification via PCR

Additionally:

• New assembly methods

• Existing reference genomes

[adapted from Shendure and Ji, 2008; Ansorge, 2009; Metzker, 2010]


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Sequencing machine in “handy” format also for smaller laboratories

2. Generation: Illumina (MiSeq, HiSeq, NextSeq)


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Background: Illumina Technologie


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100 MICRONS

A C G T

> 100 MILLION CLUSTERS PER FLOW CELL

20 MICRONS



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Background: Comparison Sanger vs Illumina

[adapted from Shendure and Ji, 2008; Ansorge, 2009; Metzker, 2010]


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Short Reads

• Good for detecting variants (single nucleotide polymorphisms /

SNPs) compared to closely-related reference genomes

• Repeat Elements, e.g. IS elements

• Phages

• Assembly (putting it back together) difficult and with gaps

• Plasmids are often mosaic and fluid in structure

Background: Problems of shot-gun data


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ABI Prism 3700

• 2. Generation – ”Next-generation” sequencing

Illumina MiSeq/HiSeq

454 (✝), IonTorrent, Qiagen GeneReader

Massive, parallel Shot-gun sequencing

• 3. Generation

PacBio RSII

Oxford Nanopore Technologies MinION

Long reads, single molecule



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Pacific Biosciences

Sequencing via polymerases attached to bottom of well

Advantages:

• Long reads for de novo assembly

Disadvantages:

• Large footprint

• High error rate – But repeated circular reading of molecule reduces

this

• Limited throughput, high costs

3. Generation: PacBio


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Oxford Nanopore Technologies (ONT)

Reading of sequence of a single molecule passing through a pore

– electric signal

Advantage:

Small & handy, e.g. use ”in the field” – Ebola

Disadvantage:

Not fully mature yet, currently under constant development

Error rate – electric signal reads “words” (kmer) of length

depends on model, homopolymers problematic

3. Generation: Nanopore


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Background: Comparison Sanger, Illumina vs Nanopore

[adapted from Shendure and Ji, 2008; Ansorge, 2009; Metzker, 2010; Goodwin, McPherson and McCombie 2016] © ESCMID eLibrary b

y author

Background: Comparison sequencers

Sanger Illumina PacBio ONT

Read length 500-1000bp 150-250 (PE) ~20,000bp <200,000bp

Throughput 0.0003Gb 4.5-5.1Gb 500Mb-1Gb <1.5Gb

Samples / run 96/384 20-24 (96) 1 1-6

Runtime 1-2h 24h 4h 48h (realtime Analysis possible)

Error profile 0.1%, Substitution

13% single pass, <1% circular consensus

~12%, Indel

Cost instrument $100,000 $695,000 $1,000

cost/Gb $212 $1,000 $750

[adapted from Goodwin, McPherson, and McCombie, 2016]


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Mapping

Comparison to reference genome

Identification of SNPs (Mutations)

Phylogenetic analysis

Assembly

De novo reconstruction / no reference

Analysis of what is not in the reference (Antibiotic resistance

genes, virulence factors, plasmids, phages,…)

Genome Analysis


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basic principles of whole genome sequencing

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