It is good practice to prepare a concentrated stock of the chromosomal DNA, as it is always easier to dilute the DNA than to concentrate it. Preparation of good quality, clean chromosomal DNA is required for efficient DNA library construction.

This end product, DNA library, is then purified and is now ready to be loaded onto the Illumina GA-II sequencer. Using previously designed PCR oligos, the adapter-modified fragments will then be enriched using PCR. This fragmented DNA will then be blunt-ended in the second step, followed by the addition of a 3′ adenine, which will then be used to ligate adapter fragments. With a starting material of good quality, clean chromosomal DNA, the user will fragment the DNA using a nebulizer, into a broad range of fragment sizes, with the majority of the fragments between 200 to 800 bp. Each section results in a sample that is then purified and carried over as the starting material in the subsequent section. Subsequent use of alternate tags (up to 12) in individual reactions will allow the user to perform multiplexed paired-end sequencing to further reduce sequencing costs and increase efficiency. The following protocol is adapted from the protocol used by the Illumina GA-II deep sequencer and is designed to help generate a bacterial DNA library that is labeled with one indexed tag. Most laboratories already carry all the components necessary for DNA library construction and would only need to purchase a few of the commercially available reagents and oligos. This is especially useful for those that are conducting deep sequencing on a small number of samples with a limited budget. 2010), all at a relatively short turn-around time and significantly reduced costs.Īlthough there are commercially available services that can provide the deep sequencing data for varying fees, one can avoid these unnecessary costs by preparing your own DNA libraries that only need to be sequenced. 2008)with the appropriate analysis software( Blankenberg et al. 2010)to shed insight into unexplainable phenotypes, but also provide sequence data to help establish evolutionary drift between or across related strains( Sapp 2007 Brinkhoff et al. 2010 Rounsley and Last 2010 Szpara et al. With the development of automation and high-throughput machines, such as the Illumina GA-II, deep sequencing can not only be used to help identify previously unknown genetic differences ( Bentley et al. 1995), whole genome sequencing (deep sequencing) has become an increasingly popular tool ( Borman et al. 1977a)and the first bacterial genome in 1995 ( Fleischmann et al. With the sequencing of the first bacteriophage genome in 1977( Sanger et al. Important base mutations, sequence deletions or repeats, and even sequence insertions, can go unnoticed from routine assays and have broader implications than one may realize. Proper identification of new strains and isolates, including important clinical isolates, often requires more than just phenotypic characterization.

T4 dna polymerase klenow fragment