Presentation on theme: "Virus discovery-454 sequencing Michel de Vries Laboratory of Experimental Virology."— Presentation transcript:
Virus discovery-454 sequencing Michel de Vries Laboratory of Experimental Virology
Introduction In 5-40 % of hospitalized patients with suspected respiratory viral infection no agent is identified. Possible problem: New or unusual subtype. New or unusual subtype. Virus Discovery cDNA-AFLP (VIDISCA) was developed in * 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
VIDISCA Fragment separation and isolation Cloning in TA vector Colony-PCR Sequencing of colony-PCR products Selective PCR (16 primer combin.)
Problems VIDISCA amplifies background ribosomal RNA (rRNA) and chromosomal DNA together with viral sequences. This background amplification interferes with VIDISCA by acting as competitors. VIDISCA can only amplify high viral load samples and/or low background samples. Clinical samples such as nose washing are full with background rRNA and chromosomal DNA.
Clinical samples 12 clinical samples (nose washings) supplied by clinical virology. Virus in the samples were identified via multiplex PCR but given double blind. Samples containing high/medium/low viral load of known viruses. For 1 out of 12 samples a viral sequences was obtained (HCoV-229E). In most samples only background rRNA and DNA was identified
Solution If a high number of fragments are randomly sequenced, a minority population can be identified.
Next Generation Sequencing One of NGS is the 454 sequencer of Roche, which is present in the AMC. Per run a maximum of 1.5 E6 beads can be used resulting in about 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. Primer A MID DNA fragment Primer B
VIDISCA- 454 sequencing Fragment separation and isolation Cloning in TA vector Colony-PCR Sequencing of colony-PCR products Selective PCR (16 primer combin.)
VIDISCA-454 Can we sequence viruses with VIDISCA-454 ? Can we use 12 MIDs in one run ?
Test with human coxsackievirus B4 12 times coxsackievirus B4 supernatant (2.0 E8 copies/ml) as input. Each with a specific MID-primer A anchor. Sequences were separated per MID, aligned and compared to GenBank database.
Result MID Nr. of sequences Viral sequences % of total Total
Result 2 Standard VIDISCAVIDISCA-454 Number of fragments Total number of viral genome nucleotides Sequenced % of viral genome. 3659
Conclusion VIDSICA-454 works!! 12 MIDs can be used, so 12 samples can be pooled in a sequence run.
Conclusion VIDSICA-454 works!! 12 MIDs can be used, so 12 samples can be pooled in a sequence run Are we ready for clinical samples??
Result 3 Again the12 clinical samples tested with VIDSICA
Result 4 In 6 out of 12 samples a virus could be identified. VIDISCAVIDISCA-454 Samples tested 12 Nr. of virus indentified 16 Viruses identified HCoV 229E HCoV OC43 RSV (2X) Rhinovirus hMPV
Conclusion With VIDISCA out of 12 clinical samples could be identified compared to 1 out of 12 with normal VIDISCA. An increase of sequence data by 454 sequencing results in a high chance of viral detection in clinical samples. We are ready for clinical samples!!
Advantage VIDISCA-454 Cheaper per sample. More samples per time unit. More sequence information per sample. Less interference by background amplification. More sensitive.
Problems One of the critical steps is measuring/calculating the amount of DNA. DNA is measured in ng/µl via fluormeter and DNA size is estimated via agarose gel. Analyses takes 1 month. Codoncode (aligning program) can not handle > 2000 sequences. Therefore you have to align in batches. Codoncode (aligning program) can not handle > 2000 sequences. Therefore you have to align in batches. For analyses via Blast sequences have to be exported in batches of 100 and then submitted. For analyses via Blast sequences have to be exported in batches of 100 and then submitted.
Acknowledgements Laboratory of experimental virology Nuno Faria Marta Canuti Martin Deijs Maarten F. Jebbink Lia van der Hoek Department of Neurogenetics Marja jakobs Frank Baas Deparment of clinical virology Richard Molenkamp Klinische Epidemiologie, Biostatistiek en Bioinformatica Barbera van Schaik Angela Luijf