1. Laboratory of Experimental Virology Virus Discovery 454 sequencing Michel de Vries micheldevries@high-throughput-sequencing.com

2. 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

3. 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.

4. Laboratory of Experimental Virology 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

5. Laboratory of Experimental Virology VIDISCA Cloning in TA vector Colony-PCR Sequencing of colony-PCR products Selective PCR (16 primer combin.)

6. Laboratory of Experimental Virology VIDISCA-454 Cloning in TA vector Colony-PCR Sequencing of colony-PCR products Selective PCR (16 primer combin.) fragmentSize (nu) 1120-200 2200-300 3300-400 - Fragment separation and isolation 454 sequencing

7. Laboratory of Experimental Virology 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.

8. Laboratory of Experimental Virology Result MIDNr. of sequencesViral sequences% of total 12805247788.3 23890328284.4 32369200784.7 42782253491.1 53056254083.1 62271200088.1 71429135494.8 83830296177.3 93018282193.5 103040284093.4 115216499195.7 122891274094.8 Total365973254788.9

9. Laboratory of Experimental Virology 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.

10. Laboratory of Experimental Virology Result 3  12 clinical samples tested with VIDISCA and VIDSICA-454 VIDISCAVIDISCA-454 Samples tested 12 Nr. of virus indentified 16 Viruses identified HCoV 229E HCoV OC43 RSV (2X) Rhinovirus hMPV

11. Laboratory of Experimental Virology Sample IDVIDISCA VIDISCA-454 (% of sequences) result by multiplex (viral copies/100 μl) 20752434NegativeNegative (<0.014 %) RSV (1.1 E6) 20752437HCoV 229E (1.1 %) HCoV 229E (4.3 E5) 20752240Negative hMPV (0.006 %) hMPV (7.6 E6) 20750951Negative HCoV OC43 (0.006%) HCoV OC43 (5.8 E4) 20751197Negative (< 0.007) HRV (2.3 E11) 20751967Negative RSV (0.33 %) RSV (3.4 E8) 20752218Negative (<0.04 %) hMPV (5.4 E4) 20752090Negative (<0.05%) HRV (8.3 E5) 20751802Negative HRV-C (0.017 %) HRV (5.6 E11) 20752358Negative (<0.012%) hMPV (2.3 E9) 20752481Negative (<0.021 %) RSV (3.3 E5) 20752346NegativeRSV (0.166 %) RSV (8.3 E7) Result 4

12. Laboratory of Experimental Virology 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.

13. Laboratory of Experimental Virology Advantage High throughput sequencing  Cost per sample are reduced by 50 %.  More samples per time unit.  More sequence information per sample.  More sensitive.

14. Laboratory of Experimental Virology Acknowledgements  Laboratory of Experimental Virology Nuno Faria Martin Deijs Maarten F. Jebbink Lia van der Hoek  Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano Marta Canuti  Department of Neurogenetics Marja Jakobs Frank Baas  Laboratory of Clinical Virology Richard Molenkamp  Department of Clinical Epidemiology, Biostatistics and Bioinformatics Barbera van Schaik Angela Luijf micheldevries@high-throughput-sequencing.com