I’m starting a new series of short tutorials. In a selfish way, these posts are for me – a vehicle for me to clarify my own comprehension of a given topic. I might also tell you about a solution to a problem I have been troubleshooting.
The first post in this series is about fosmids. There seems to be some public confusion as to what they are or what they can be used for – basically, I have been confused. Apparently I had forgotten my microbiology coursework along the way. Many genome sequencing projects – such as the human genome project – have utilized fosmids to create libraries prior to sequencing, but it wasn’t until hearing about JGI’s fungal and metagenomic sequencing initiatives did I hear the term fosmid mentioned frequently.
Fosmids are used when preparing genomic libraries for genome sequencing. Fosmids are circular DNA of bacterial origin – technically plasmids – but where typical plasmids exist in high copy number (up to 100 copies per cell) and possess small (3 to 6 kb) inserts, fosmids are present as a single copy in a cell and may possess inserts upwards of 40 kb. Fosmids are advantageous because they produce stable libraries for genome sequencing. They have a tendency to provide fairly uniform coverage, so they are optimal for closing gaps in whole genome alignments. In addition to genome sequencing, they have also been used for metagenomics and expression studies.
Fosmids are derived from the fertility plasmid (or F-plasmid) and are responsible for the formation of the sex pilus during bacterial conjugation. This plasmid contains both origin and partitioning genes derived from the F’-episome and as a result, the plasmid is kept as a single copy clone, which comes in handy during genomic DNA library construction. Fosmid vectors are derived from random shearing – which yields more uniform coverage when comparing against other library cloning methods.
Cosmids may also be useful for genome sequencing projects, but unlike fosmids, they are multi-copy vectors that are generally present at anywhere from 20-70 copies per cell and this high copy number leads to instability and lost segments of genomic DNA. This can be an issue for closing gaps in genome alignment, but if you’ve got high sequencing depth and a small genome to sequence, it may not be much of an issue. Most importantly, with high copy number plasmids, such as cosmids, the chance of recombination increases which can disrupt and rearrange genomic DNA inserts prior to sequencing.
Lastly, fosmids can be useful for chromosome specific sequencing and as cytological markers for chromosome identification. The image above — which comes from this paper — shows the identification of chloroplast genome isolation and sequencing from fosmids; a similar technique can be used to isolate and sequence specific chromosomes. Also, fosmids may be used as cytological markers with in situ hybridization on metaphase karyotypes and sorted using flow cytometric methods.