An algorithm for the determination and quantification of components of nucleic acid mixtures based on single sequencing reactions

We describe an algorithm that is based on a set of linear equations which can be solved when the sequencing profiles of the individual components are known and when the number of sequenced nucleotides is larger than the number of components in the mixture. We have implemented the procedure for one type of sequencing approach, pyrosequencing, which produces a stepwise output of peaks that is particularly suitable for the procedure. As an example we use signature sequences from ribosomal RNA to distinguish and quantify several different species in a mixture. Using simulations, we show that the procedure may also be applicable for dideoxy sequencing on capillary sequencers, requiring only some instrument specific adaptations of protocols and software.The parallel sequencing approach described here may become a simple and cheap alternative to microarray experiments which aim at routine re-determination and quantification of known nucleic acid components from environmental samples or tissue samples.The identification of individual components from a mixture of nucleic acid sequences relies currently on molecular hybridization approaches, such as microarrays. Technically, these make use of the inherent combinatorial complexity of strings of nucleotides, as well as the base pairing reaction between single stranded molecules. These allow to potentially resolve vast numbers of different sequences, provided one knows which sequences can occur in the mixture. In contrast, DNA sequencing reactions do not require a priori knowledge of the sequence that is to be determined, but they can only identify one sequence at a time. However, the patterns produced in sequencing reactions have also an inherent combinatorial complexity with respect to the order and height of peaks. Accordingly, mixtures of sequencing reactions should also produce characteristic patterns which reflect the components of the mixture. Thus, it should be possible to deconvolute a mixture pattern into its individua