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24,25 Its main difference from the classic Sanger sequencing is that pyrosequencing relies on the detection of pyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides. The release of pyrophosphate is conveyed [http://en.wikipedia.org/wiki/SAR1B SAR1B] into light using enzyme reactions, which is then converted into actual sequence information.23 In the initial years of high-throughput sequencing, scientists embraced the new technology and hence discovered the existence of the ��rare biosphere��.26 However, in many cases the apparent assignment of a microbial operational taxonomic unit (OTU) was in fact an attribute of sequencing errors, which caused an overinflation of the diversity estimates.27 Noise generated by this 454 pyrosequencing technology affected different aspects of metagenomic data analysis and led to biased results.28 PCR errors may lead to replicate sequence artifacts, which can cause overestimation of species abundance and functional gene abundance in 16S rRNA and full shotgun metagenomics, respectively. PCR can also generate noise in the form of single base pair errors (ie, substitutions, deletions) that can cause frame shifts for protein coding genes in shotgun meta-genomics. Moreover, PCR chimeras (sequences generated by undesired end-joining of two or more true sequences) can also affect 16S metagenomics [http://www.selleckchem.com/products/Dasatinib.html selleck chemical] results with respect to species distribution.29 Sequencing errors can also occur due to the actual chemistry underlining the technology. For example, there is an inherent difficulty in clearly identifying the intensity of 454 pyrosequencing-generated flowgrams. This task becomes even more difficult during the sequencing of homopolymers.30 The 454 pyrosequencing technology can generate reads up to 1,000 bp in length and ~1,000,000 reads per run. The relatively long read length generated by this technology (in comparison to other sequencing technologies) allows a significantly less error-prone assembly in shotgun metagenomics and permits [http://www.selleckchem.com/products/s-gsk1349572.html GSK1349572 ic50] greater annotation accuracy.31,32 The cost of sequencing using 454 pyrosequencing technology is estimated at around US$20 per Mb, but it has a relatively low coverage of 0.7 GB per sequencing run. With respect to pyrosequencing,