Presentation on theme: "Virus discovery-454 sequencing"— Presentation transcript:
1Virus discovery-454 sequencing Michel de VriesLaboratory ofExperimental Virology
2IntroductionIn 5-40 % of hospitalized patients with suspected respiratory viral infection no agent is identified.Possible problem: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 targetsequence.Based on restriction enzyme cleavage sites + ligation of adaptors + PCR.* van der Hoek et al, Nature medicine 2004
3VIDISCA Selective PCR (16 primer combin.) Fragment separation and isolationCloning in TA vectorColony-PCRSequencing of colony-PCR productsSelective PCR (16 primer combin.)
4ProblemsVIDISCA 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.
5Clinical samples12 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
6Solution If a high number of fragments are randomly sequenced, a minority population can be identified.
7Next 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
8VIDISCA- 454 454 sequencing Selective PCR (16 primer combin.) Fragment separationand isolationCloning in TA vectorColony-PCRSequencing of colony-PCR productsSelective PCR (16 primer combin.)VIDISCA-454454sequencing
9VIDISCA-454 Can we sequence viruses with VIDISCA-454 ? Can we use 12 MIDs in one run ?
10Test 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.
11Result MID Nr. of sequences Viral sequences % of total 1 2805 2477 88.323890328284.432369200784.742782253491.153056254083.162271200088.171429135494.883830296177.393018282193.5103040284093.4115216499195.71228912740Total365973254788.9
12Result 2 Standard VIDISCA VIDISCA-454 Number of fragments. 11 35 Total number of viralgenome nucleotidesSequenced.26464365% of viral genome.3659
13Conclusion VIDSICA-454 works!! 12 MIDs can be used, so 12 samples can be pooled in a sequence run.
14Are we ready for clinical samples?? ConclusionVIDSICA-454 works!!12 MIDs can be used, so 12 samples can be pooled in a sequence runAre we ready for clinical samples??
15Result 3 Again the12 clinical samples tested with VIDSICA-454
16In 6 out of 12 samples a virus could be identified. Result 4In 6 out of 12 samples a virus could be identified.VIDISCAVIDISCA-454Samples tested12Nr. of virusindentified16Viruses identifiedHCoV 229EHCoV OC43RSV (2X)RhinovirushMPV
18Conclusion We are ready for clinical samples!! 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!!
19Advantage VIDISCA-454 More sensitive. Cheaper per sample. More samples per time unit.More sequence information per sample.Less interference by background amplification.More sensitive.
20ProblemsOne 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.For analyses via Blast sequences have to be exported in batches of 100 and then submitted.
21Acknowledgements Laboratory of experimental virology Nuno FariaMarta CanutiMartin DeijsMaarten F. JebbinkLia van der HoekDepartment of NeurogeneticsMarja jakobsFrank BaasDeparment of clinical virologyRichard MolenkampKlinische Epidemiologie, Biostatistiek en BioinformaticaBarbera van SchaikAngela Luijf