deletions | additions       diff --git a/Library Preparation and Sequencing .md b/Library Preparation and Sequencing .md  index 9da70ae..94a37d7 100644  --- a/Library Preparation and Sequencing .md  +++ b/Library Preparation and Sequencing .md 
...
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 300 bp  (PE300) reads on the Illumina MiSeq, we shoot for a fragment size (including adapters) of 600-900bp. 600-900 bp.  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 3.5 Mb  genome. On the HiSeq with PE125bp PE125 bp  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. ##Collaborate  As described above, current Illumina sequencing systems have much greater capacity than is needed for sequencing a single genome. This means it can be generally beneficial to combine many samples into a single run of a machine. Unfortunately, our experience has been that sequencing facilities will typically not help in the coordination of such pooling of samples (we assume because they do not want to oversee the pooling or deal with the associated accounting hassles). Therefore this means it is up to the users to carry out such coordination. Though this can sometimes be complicated it is generally worth it since one can pool together many genomes or metagenomes into single runs of a system and still get enough data for each project, thus making the sequencing cost per project significantly lower. For this to work well, what one needs to do is to coordinate the use of barcodes to tag each sample, coordination of the pooling, and some informatics work at the end to "demultiplex" samples from each other.