RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms.It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile.Detection and identification of bacteria directly from clinical samples by broad-range PCR targeting the 16S rRNA gene and DNA sequencing (direct 16S rRNA gene sequencing) make up a well-established method in many laboratories. This method gives the possibility to identify bacteria that died during transportation or as a consequence of antibiotic treatment and to uncover bacteria with special growth requirements. The latest advances in PCR and sequencing technology also offer a more rapid identification than that obtained with standard phenotypic methods that depend on bacterial growth.Because of difficulties in the interpretation of DNA chromatograms resulting from direct sequencing of polybacterial samples, the use of this diagnostic tool has been limited to infections that are predominantly monobacterial. We earlier described RipSeq (iSentio, Bergen, Norway), a web-based application for the analysis of mixed DNA chromatograms (12). In the same article, we presented the RipSeq performance on a number of mixed DNA chromatograms obtained by direct sequencing from saline suspensions containing two and three different bacterial species and discussed the possible benefits and limitations one could experience if the method was to be applied to human clinical samples. In this study, direct 16S rRNA gene sequencing was used to investigate 264 human clinical samples from a wide range of locations, including typical polybacterial specimens such as abscesses and pleural fluids. All mixed DNA chromatograms were analyzed with the RipSeq program, and the sequence-based results were compared to routine culture-based diagnostics in our hospital laboratory.We also discuss special concerns in the selection of a lysis procedure and primers for use on polymicrobial samples as well as the establishment of a reliable negative control.