Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.

polymerase, ATP sulfurylase, firefly luw ciferase, and a nucleotide-degrading enzyme (such as apyrase). Repeated cycles of deoxynucleotide addition are performed. A A Sequencing Method Based On deoxynucleotide d l incorporated into the growing DNA strand if it is com-Real-Time Pyrophosphate plementary strand. The synthesis to the base of DNA in the is accompa-template ---Mostafa Ronaghi, Mathias UhlCn, and Pi1 NyrCn* nied by releak of PPi equal in molarit; to that of the incorporated deoxynucleotide.

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A cDNA encoding the precursor of the bovine mitochondrial phosphate carrier protein has been cloned from a bovine cDNA library using a mixture of 128 different 17-mer oligonucleotides as hybridisation probe. The protein has an N-terminal extension of 49 amino acids not present in the mature protein. This extension has a net positive charge and is presumed to direct the import of the protein from the cytoplasm to the mitochondrion. Comparison of the protein sequence of the mature phosphate carrier with itself, with ADP/ATP translocase and with the uncoupling protein from brown fat mitochondria shows that all three proteins contain a 3-fold repeated sequence -100 amino acids in length, and that the repeats in the three proteins are related to each other. This inplies that the three proteins have related threedimensional structures and mechaniims and that they share a common evolutionary origin. The distribution of hydrophobic residues in the phosphate carrier protein suggests that each repeated 100 amino acid element is composed of two membrane-spanning a-helices linked by an extensive hydrophilic domain. This model is similar to that first proposed for the ADP/ATP translocase and later for the brown fat mitochondria uncoupling protein.