The second problem, which is mainly encountered in morphological analysis, is species-level identification, which is often impossible to achieve in larval samples. Our results show the effectiveness of 454 pyrosequencing for the analysis of an engineered mixture of adult insects. We were able to gain species-level resolution for all abundant species in the mixture. However, 454 pyrosequencing missed 6 low abundance species but provided DNA sequence evidence for the presence of 9 other species undetected in single specimen Sangerbased analysis. What might appear to be a puzzling result can be explained as a consequence of two issues. The first issue, which can explain failure in identification of 6 lower abundance species, is the bias associated with binding PCR primers to targettemplate DNA in a mixture. Species with higher affinity in their primer binding sites and/or species with higher abundance can capture more primer molecules during the process of PCR annealing. Consequently, species with lower affinity to primers and/or lower abundance may not yield amplicons. Deeper next-generation sequencing can potentially alleviate this issue. Other studies, especially studies targeting rare HIV mutants have used this strategy to detect low abundance virus genes in clinical samples. The second issue, which can explain the detection of DNA sequences from 9 species that were not originally present in the specimens we pooled in this analysis, is likely the result of carryover of DNA from these 9 species from the liquid preservation media to the bodies of specimens selected for the pooling experiment. The authors have recently shown that DNA from specimens can be detected directly from preservative ethanol. In addition, we have been able to obtain DNA sequences from majority of species in a mixture by directly 454 pyrosequencing the ethanol used as preservative for bulk benthic samples. Because the total number of species detected from a single 454 pyrosequencing analysis is larger when compared to single specimen Sanger-based DNA barcoding, and yet all common species are detected in 454 analysis, we believe 454 pyrosequencing is advantageous as compared to a single specimen approach. Our work for the first time used the standard COI DNA barcode information in 454 pyrosequencing approach for the analysis of specimens from two orders of insects. Although the majority of prior studies that employed 454 pyrosequencing for biodiversity surveys have focused on ribosomal markers such as 16S rDNA and 18S rDNA we decided to use COI firstly because a small mini-barcode sequence of this gene allows species-level resolution in most animal and protist groups tested and second, it can be linked to an expanding DNA barcode reference library. Lack of universal primers has been used as an argument against the use of this gene region in next-generation sequencing analysis of environmental samples. However, our results show that COI PCR primers can be effective in amplifying multiple templates. The bias associated to COI is comparable to reported bias associated with other genes. This bias can obscure quantitative analysis of species abundance and can also negatively influence the detection of low abundance species when sequencing depth is not maximized. Nevertheless, our analysis shows that the percentage of sequence reads obtained in environmental barcoding using 454 pyrosequencing is comparable to the abundance measure obtained through counting individuals in the bulk sample. This is perhaps due to the fact that the relative abundance of species in nature can potentially offset any bias from primer-binding in PCR.