Laboratory of Experimental Virology

Virus Discovery 454 sequencing

Michel de Vries

micheldevries@high-throughput-sequencing.com

Laboratory of Experimental Virology

Introduction

In 5-40 % of hospitalized patients with suspected respiratory viral infection no agent is identified.

Possible problem: New or variant.

• Virus Discovery cDNA-AFLP (VIDISCA) was developed in 2004. *

• VIDISCA can amplify both RNA and DNA viruses without prior knowledge of the target sequence.

• Based on restriction enzyme cleavage sites + ligation of adaptors + PCR.

* van der Hoek et al, Nature medicine 2004

Laboratory of Experimental Virology

Problems

VIDISCA amplifies background ribosomal RNA (rRNA) and chromosomal DNA

together with viral sequences.

This background amplification interferes with VIDISCA by acting as competitors.

Clinical samples such as nose washing are full with background rRNA and

chromosomal DNA.

Solution

If a high number of fragments are randomly sequenced, a

minority population can be identified.

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High throughput sequencing

High throughput sequencing makes it possible to sequence a large amount of DNA fragments in a single run.

One of these machines is the 454 FLX sequencer of Roche.

The method binds a single DNA fragment to a bead which is clonally amplified.

Per run a maximum of 1.5 million beads can be used resulting in about 400.000 quality sequences.

Multiplex identifiers (MID) are 10 nucleotides long barcodes that are recognized by our software.

The MID can be incorporated into samples allowing multiple samples to be pooled.

By using MIDs more samples can be processed in a single run

Laboratory of Experimental Virology

VIDISCAVIDISCA

Cloning in TA vector

Colony-PCR

Sequencing of colony-PCR products

Selective PCR (16 primer combin.)

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VIDISCA-454

Cloning in TA vector

Colony-PCR

Sequencing of colony-PCR products

Selective PCR (16 primer combin.)

fragment Size (nu)

1 120-200

2 200-300

3 300-400

-Fragment separation and isolation

454 sequencing

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Virus culture supernatant in VIDSICA-454

12 times coxsackievirus B4 supernatant (2.0 E8 copies/ml) as input.

Each with a specific MID-primer A anchor.

Sequences per MID were separated, aligned and compared to GenBank database.

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Result

MID Nr. of sequences Viral sequences % of total

1 2805 2477 88.3

2 3890 3282 84.4

3 2369 2007 84.7

4 2782 2534 91.1

5 3056 2540 83.1

6 2271 2000 88.1

7 1429 1354 94.8

8 3830 2961 77.3

9 3018 2821 93.5

10 3040 2840 93.4

11 5216 4991 95.7

12 2891 2740 94.8

Total 36597 32547 88.9

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Clinical samples in VIDSICA-454

12 clinical samples (nose washings).

Again each sample with a specific MID-primer A anchor

Virus in the samples were previously identified via multiplex PCR but given double blind.

Samples containing high/medium/low viral load of known viruses.

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Result 3

12 clinical samples tested with VIDISCA and VIDSICA-454

VIDISCA VIDISCA-454

Samples tested 12 12

Nr. of virus

indentified1 6

Viruses identified

HCoV 229E

HCoV 229E

HCoV OC43

RSV (2X)

Rhinovirus

hMPV

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Sample ID VIDISCA VIDISCA-454

(% of sequences)

result by multiplex

(viral copies/100 μl)

20752434 Negative Negative (<0.014 %) RSV

(1.1 E6)

20752437 HCoV 229E HCoV 229E

(1.1 %)

HCoV 229E

(4.3 E5)

20752240 Negative hMPV

(0.006 %)

hMPV

(7.6 E6)

20750951 Negative HCoV OC43

(0.006%)

HCoV OC43

(5.8 E4)

20751197 Negative Negative

(< 0.007)

HRV

(2.3 E11)

20751967 Negative RSV

(0.33 %)

RSV

(3.4 E8)

20752218 Negative Negative

(<0.04 %)

hMPV

(5.4 E4)

20752090 Negative Negative

(<0.05%)

HRV

(8.3 E5)

20751802 Negative HRV-C

(0.017 %)

HRV

(5.6 E11)

20752358 Negative Negative

(<0.012%)

hMPV

(2.3 E9)

20752481 Negative Negative

(<0.021 %)

RSV

(3.3 E5)

20752346 Negative RSV

(0.166 %)

RSV

(8.3 E7)

Result 4

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Conclusion

VIDISCA can be combined using high throughput sequencing.

Twelve MIDs can be included allowing 12 samples to be pooled.

By pooling samples we can sequence at least 48 samples in a single run.

High throughput sequencing result higher viral genome nucleotides sequenced.

VIDISCA-454 can identify viral fragments from direct patient material.

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Advantage High throughput sequencing

Cost per sample are reduced by 50 %.

More samples per time unit.

More sequence information per sample.

More sensitive.

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Acknowledgements

Laboratory of Experimental Virology

Nuno FariaMartin DeijsMaarten F. JebbinkLia van der Hoek

Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano

Marta Canuti

Department of Neurogenetics

Marja JakobsFrank Baas

Laboratory of Clinical Virology

Richard Molenkamp

Department of Clinical Epidemiology, Biostatistics and Bioinformatics

Barbera van SchaikAngela Luijf

micheldevries@high-throughput-sequencing.com