Viral genome sequencing: applications to clinical management and public health

Professor Judy Breuer

Why do whole viral genome sequencing

Genome sequencing allows detection of multigenic resistance in widely spaced genes Genome sequencing may resolve nosocomial transmission events especially for DNA viruses where there is insuffient variation in small fragments Genome sequencing may identify outbreaks or patterns of spread

Index case Samples Contacts Samples

Patient 1 Varicella vesicle (4) n/a n/a

Subject 2 Varicella vesicle (3) Patient 3 Varicella vesicle (2)

Subject 4 Zoster Vesicle (1) Patient 5 Varicella vesicle (1)

Subject 6 Varicella vesicle (1) Patient 7 Varicella vesicle (1)

Patient 8 Zoster vesicle (1) Patients 9 & 10 Varicella vesicle (1,1)

Specimen 1 Varicella vesicle (1) n/a n/a

Specimen 2 Varicella vesicle (1) n/a n/a

Specimen 3 Varicella Skin swab n/a n/a

Specimen 4 Gastric biopsy n/a n/a

Nosocomial Transmission

Clade 1

Clade 3

Clade 5

Clade 1

Clade 3

Clade 5

Transferred to ITU

8

9

10

1 2 3 4 5 6 7 9 10 11 12 14 15 25 26 27 28

Shingles diagnosed chickenpox chickenpox death

Fatal varicella in renal transplant recipients

Clade 1

Clade 3

Clade 5

clade 1

clade 2

clade 3

clade 5

clade 4

Clade 1 viruses are the most common

VZV > 99% similarity between viruses from a single clade

Problems with whole viral genome sequencing

Virus are intracellular organisms and unlike bacteria, sufficient viral nucleic acid for good quality NGS sequencing cannot easily be obtained from culture (human DNA overwhelms sequence) PCR can be used to amplify fragments for sequencing but this is not suitable for larger viruses or small volume clinical specimens (contain too little viral nucleic acid)

(6) Reference guided assembly using Burrows-Wheeler Aligner (mapped vs. VZV strain Dumas , pOka or vOKA)

(5) Quality control: Removes poor quality reads (QUASR)

(4) Sequencing (Illumina) generating paired-end reads (76bp) Incl 12-16 rounds PCR

(1) Total DNA extracted from clinical sample (± WGA)

(2) Fragmentation and library preparation ( Illumina-based protocol)

(3) Target DNA isolated by hybridisation with custom 120-mer RNA baits

Purifying viral DNA for NGS

Sample

Ratio of Viral: Human Genome copies* % on

target

reads

% Genome

coverage Mean read

depth

per base Pre-hybridisation Post-hybridisation >5-fold >100-fold

VZV

Vesicle 10299 1157666 93.69 >99% >97% 3022

CSF 34976 1006398 64.47 >99% >97% 2416

Saliva 51987 9855143 42.61 100% >98% 1096

EBV

Blood

86.37 >99% >98% 2523

Cell lysate 52.84 >98% >97% 2599

KSH

V

Cell lysate 90.97 >98% >93% 1773

Near full length genome coverage Very high per-base read depth

* Determined by qPCR quantification of ORF27 (VZV) and KRAS (Human)

100-1000 fold enrichment

High % of on target reads

Depledge et al. PLoS One, 2011

SureSelect enriches for target DNA

Evolution and pathogenesis Sequencing viruses by NGS

A C A A T

Biallelic positions- important for minority variant resistance etc

Consensus

Multiple sequence reads for each base

Method Validated

1. Highly sensitive – can recover <1000 VZV genomes in < 10ng DNA Depledge 2011

2. Reproducible and representative of the original Depledge 2013

3. Less mutagenic than PCR (without loss of population structure due to culture)

Depledge 2011

Error<1%

Targeted enrichment

*

*

*

*

Viruses from transmission cluster

Kundu, Depledge unpub

* * *

*

*

* * * *

*

*

*

*

* *

* *

* * * *

*

* * *

*

Clade 1 Clade 3 Clade 5 Clade 4 Clade 2

Kundu, Depledge unpub

Outbreak monitoring

Kundu Clin Infect Dis 2013

Kundu Clin Infect Dis 2013

Kundu Clin Infect Dis 2013

Kundu Clin Infect Dis 2013

Substitutions between A and B

Kundu Clin Infect Dis 2013

20 SNPs, (6 coding for aa substitution) 9 Informative ie biallelic

Direction of Transmission

Minority variant in B becomes fixed in A following transmission bottleneck Confirms direction of transmission is B to A

Kundu Clin Infect Dis 2013

What does this study tell us 1. Whole genome sequencing identifies nosocomial

transmission(even where patients are isolated)

2. Norovirus shed by immunocompromised is infectious (A became infected only after moving to same ward as B)

3. Norovirus infection triggers chronic diarrhoea in immunocompromised A developed diarrhoea only after becoming infected with norovirus

4. Deep sequencing data can be used to identify direction of spread

Breuer group UCL

Viral genome sequencing Dan Depledge Samit Kundu Julianne Lockwood Eleanor Grey Fanny Wegner Rachel Williams Helena Tutil Mark Quinlivan Mette Christiansson

Cambridge University

Ian Goodfellow Lucy Thorne

GOSH John Hartley

UCL Genomics Mike Hubank

PHE Portsmouth Gill Underhill