sequencing technologies with short read lengths means that much of future metagenomic data will be error-prone. Taken in combination, these factors make the assembly of metagenomic sequence reads into genomes difficult and unreliable. Misassemblies are caused by the presence of repetitive DNA sequences that make assembly especially difficult because of the difference in the relative abundance of species present in the sample. Misassemblies can also involve the combination of sequences from more than one species into chimeric contigs. There are several assembly programs, most of which can use information from paired-end tags in order to improve the accuracy of assemblies. Some programs, such as Phrap or Celera Assembler, were designed to be used to assemble single genomes but nevertheless produce good results when assembling metagenomic data sets. Other programs, such as Velvet assembler, have been optimized for the shorter reads produced by second-generation sequencing through the use of de Bruijn graphs. The use of reference genomes allows researchers to improve the assembly of the most abundant microbial species, but this approach is limited by the small subset of microbial phyla for which sequenced genomes are available. After an assembly is created, an additional challenge is "metagenomic deconvolution", or determining which sequences come from which species in the sample. Gene prediction Metagenomic analysis pipelines use two approaches in the annotation of coding regions in the assembled contigs. The first approach is to identify genes based upon homology with genes that are already publicly available in sequence databases, usually by BLAST searches. This type of approach is implemented in the program MEGAN4. The second, ab initio, uses intrinsic features of the sequence to predict coding regions based upon gene training sets from related organisms. This is the approach taken by programs such as GeneMark and GLIMMER. The main advantage of ab initio prediction is that it enables the detection of coding regions that lack homologs in the sequence databases; however, it is most accurate when there are large regions of contiguous genomic DNA available for comparison. Species diversity Gene annotations provide the "what", while measurements of species diversity provide the "who". In order to connect community composition and function in metagenomes, sequences must be binned. Binning is the process of associating a particular sequence with an organism. In similarity-based binning, methods such as BLAST are used to rapidly search for phylogenetic markers or otherwise similar sequences in existing public databases. This approach