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When growing bacteria in culture, as described in this workflow, it should almost always be possible to get enough DNA to use PCR-free TruSeq and therefore minimize library preparation biases in the genome assembly.  ##Considerations in Library Preparation  Insert size: The tradeoff with insert size is between utility for assembly (larger is better) and ability of those fragments to amplify on the Illumina flowcell for sequencing (smaller is better). The optimal fragment size also depends on the length of reads used (with longer read-lengths, longer insert sizes are useful for scaffolding). The final consideration is the amount of DNA available for sequencing. While having all inserts be exactly 750 base pairs (bp) might be ideal, such a stringent size-selection could result in the recovery of only a very small amount of DNA. In our lab, with paired end 300bp (PE300) reads on the Illumina MiSeq, we shoot for a fragment size (including adapters) of 600-900bp. Different sequencing facilities have different opinions on this topic and it is worth having a discussion with your sequencing facility's point of contact before making any libraries. It is very important that all samples have similar library sizes if multiplexing as described below.  ##Multiplexing  The capacity of an Illumina MiSeq with PE300 reads is around 15 Gigabases (Gb), which would result in a coverage of 4300X for a typical bacterium with a 3.5Mb genome. On the HiSeq with PE125bp reads, this would be over 14,000X coverage. Currently, the recommended coverage for a bacterial genome assembly is 20-200X depending on the choice of assembler. Therefore, sequencing a single bacterial genome on a full MiSeq or HiSeq run is a significant waste of money and reagents. Furthermore, some current genome assembly algorithms do not perform well given an excess of data, and require down-sampling (i.e., throwing away data) to achieve the recommended coverage for assembly. We typically multiplex 10-48 genomes on a PE300 MiSeq run and many more on a HiSeq run. If using a kit for library prep, multiplexing is quite straightforward since there are a number of barcoded adaptors that come with the kit. Demultiplexing can be performed by the sequencing facility.