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##Kit Options  Whether you chose to make libraries yourself, or use a provider the next major choice is of the type of kit. The two major different choices with Illumina kits are the Nextera transposase-based kits or the TruSeq kits (with or without PCR). These kits are available from Illumina, but there are also comparable options from other vendors (e.g. New England Biolabs). The pros and cons of each type of kit are listed below:  + Nextera: Pro – Can use It allows for  very low amounts of input DNA, down to 1ng in the case of the Nextera XT kit. Con – the transposase has an insertion bias and the extensive PCR required for low input samples will also impact the final assembly. + TruSeq (our recommendation): Pro – If the The  PCR-free protocolis used this  minimizes library  bias due to fragmentation (by by  using mechanical shearing) instead of enzymatic DNA fragmentation,  and by eliminating  PCR, resulting in better assemblies. Con – requires a large amount of DNA (1\(\mu\)g (at least 1\(\mu\)g  for PCR-free). There is also now a TruSeq LT kit which only requires 100ng of DNA but does entail some PCR so may provide a middle option between PCR-free TruSeq and Nextera. 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 (the (with  longer the read, the read-lengths,  longerof an  insert size is needed sizes are required  for scaffolding). The final consideration is the amount of DNA available for sequencing. While having all inserts be exactly 750bp would be handy, it ideal, such a stringent size-selection  would be extremely difficult to get enough DNA to sequence. result in the recovery of only a very small amount of DNA.  In our lab, with paired end 300bp reads on the Illumina MiSeq, we shoot for an insert size range of 600-900 bp. 600-900bp.  Different sequencing facilities have different opinions on this topic and it is worth having this discussion before making libraries. ##Multiplexing  The capacity of an Ilumina MiSeq with PE300bp reads is around 15 Gb which would result in a coverage of 4300X for a typical bacterium with a 3.5MB genome. On the HiSeq with PE 125bp reads this would be over 14,000X coverage. Typically the recommended coverage for a bacterial genome assembly is 30-100X depending on the choice of assembler. Therefore, sequencing a single bacterial genome on a run is a significant waste of money and reagents. Furthermore many assemblers do worse with too much data, requiring downsampling. We typically multiplex 10-20 genomes on a PE300bp 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.