Throughput sequencing setting, by combining VIDISCA with Roche 454 GS FLX Titanium sequencing. Nowadays molecular techniques are becoming the standard for the discovery of new viruses. Some methods use a conserved region for universal primer design, based on the known viral genomes. These methods are applicable to specific virus families, but cannot be used for all viruses. Furthermore, some yet unknown viruses could be too diverse and therefore remain negative in these kind of detection techniques. Sequence independent amplification methods, such as VIDISCA and random-PCR, can identify viral sequences without prior knowledge of a viral genome. Unfortunately, the detection of unknown viral pathogens in respiratory clinical material is difficult with these sequence independent virus discovery methods because of low viral load and high background nucleic acids in these samples. During the last years sequence independent virus discovery techniques were mostly used with virus BKM120 culture supernatant, as they contain high concentrations of viral genomes, or to discover previously unknown DNA viruses. So far no study has been able to identify novel human respiratory RNA viruses with sequence independent amplification techniques. Thus sequence independent amplification techniques like VIDISCA have to be optimized to allow discovery without requiring a culture amplification step. We managed to unfavor rRNA amplification by adjusting the reverse transcription step. Utilization of primers during cDNA synthesis that poorly recognize rRNA, in combination with the addition of oligo’s that halt cDNA synthesis on rRNA templates successfully decreased interfering background amplification. Additionally, using a single restriction enzyme with low numbers of recognition sites in 28S rRNA provided further reduction of useless and interfering amplification. Thus all steps increased the ratio of viral genome versus rRNA amplifications, and the benefit was shown in VIDISCA-high throughput sequencing of clinical samples containing known viruses. In the majority of clinical samples the virus was easily identified by VIDISCA-454. In two cases even an input of 140 and 190 genome copies of an adenovirus and influenza A virus could be detected by VIDISCA-454. Ideally, oldprotocol VIDISCA-454 should have been compared with optimized VIDISCA-454. However, this comparison is regrettably not possible due to limitation of the respiratory clinical specimens that we used. Thus we rely on all the reconstructions and monitoring performed with normal VIDISCA. As mentioned above, the use of one restriction enzyme diminished background rRNA amplification. There is one additional advantage of single restriction enzyme usage. In the traditional VIDISCA two restriction enzymes were combined and only fragments that have one restriction site on the 59 site and the other in the 39 site are amplified after ligation. Such VIDISCA amplification is restricted in case one of the two enzymes has few recognition sites optimal.