48
A. Meyerdierks and F.O. Glöckner
DNA Isolation
DNA Purification
- liquid based (cosmid, fosmid)
- matrix based (BAC)
Enzymatic Manipulation
- blunt ending (fosmid)
- partial digest (cosmid, BAC)
Size Selection [2x]
- PFGE (Fosmid, BAC)
- [one dimensional agarose
gel electrophoresis
(Fosmid, Cosmid)]
Ligation
Transformation
- in vitro packaging&infection
(fosmid, cosmid)
- electroporation (BAC)
Environmental Sample
[Enrichment]
Library Storage
- arrays
- pools
Fig. 2.3 Overview of the construction of large insert metagenomic libraries
single recombinant (e.g. average insert size/genome size) (Sambrook and Russel
2001). In metagenome analysis, several additional factors have to be considered.
The size of a metagenomic library that is needed to be representative for an environmental sample, or that contains statistically one clone carrying a marker gene
of interest is difficult to calculate. Among the additional factors that have to be
taken into account are the diversity and composition of the microbial community,
and the genome sizes of the different microorganisms in the sample. The average genome size, calculated for the 815 bacterial and archaeal genomes listed on
the homepage of the National Center for Biotechnology Information (September
2009; http://www.ncbi.nlm.nih.gov/) is about 3.2 Mbp. Nevertheless, genome size
can vary considerably. Currently the two extremes are the genomes of Candidatus
Carsonella ruddii (159,662 bp), an endosymbiotic gammaproteobacterium, and
Sorangium cellulosum “So ce 56” (13 Mbp), a soil bacterium belonging to the
Myxobacteria. In addition, incomplete lysis of cells from complex environmental
samples may result in a biased library. Repetitive elements and an extreme G+C
content may also introduce a bias (Abulencia et al. 2006) as well as the presence of
toxic gene products such as phage genes (Edwards and Rohwer 2005).
2.3.5.4 Storage of Metagenomic Libraries
Metagenomic libraries may be stored at several different stages. If the library cannot be plated at once, the ligation or the packaged phage heads can be stored at
–80 ◦ C, after shock freezing. Prior to deep freezing, the phage solution has to be supplemented with DMSO (Sambrook and Russel 2001, Promega Corporation 2007)
A. Meyerdierks and F.O. Glöckner
DNA Isolation
DNA Purification
- liquid based (cosmid, fosmid)
- matrix based (BAC)
Enzymatic Manipulation
- blunt ending (fosmid)
- partial digest (cosmid, BAC)
Size Selection [2x]
- PFGE (Fosmid, BAC)
- [one dimensional agarose
gel electrophoresis
(Fosmid, Cosmid)]
Ligation
Transformation
- in vitro packaging&infection
(fosmid, cosmid)
- electroporation (BAC)
Environmental Sample
[Enrichment]
Library Storage
- arrays
- pools
Fig. 2.3 Overview of the construction of large insert metagenomic libraries
single recombinant (e.g. average insert size/genome size) (Sambrook and Russel
2001). In metagenome analysis, several additional factors have to be considered.
The size of a metagenomic library that is needed to be representative for an environmental sample, or that contains statistically one clone carrying a marker gene
of interest is difficult to calculate. Among the additional factors that have to be
taken into account are the diversity and composition of the microbial community,
and the genome sizes of the different microorganisms in the sample. The average genome size, calculated for the 815 bacterial and archaeal genomes listed on
the homepage of the National Center for Biotechnology Information (September
2009; http://www.ncbi.nlm.nih.gov/) is about 3.2 Mbp. Nevertheless, genome size
can vary considerably. Currently the two extremes are the genomes of Candidatus
Carsonella ruddii (159,662 bp), an endosymbiotic gammaproteobacterium, and
Sorangium cellulosum “So ce 56” (13 Mbp), a soil bacterium belonging to the
Myxobacteria. In addition, incomplete lysis of cells from complex environmental
samples may result in a biased library. Repetitive elements and an extreme G+C
content may also introduce a bias (Abulencia et al. 2006) as well as the presence of
toxic gene products such as phage genes (Edwards and Rohwer 2005).
2.3.5.4 Storage of Metagenomic Libraries
Metagenomic libraries may be stored at several different stages. If the library cannot be plated at once, the ligation or the packaged phage heads can be stored at
–80 ◦ C, after shock freezing. Prior to deep freezing, the phage solution has to be supplemented with DMSO (Sambrook and Russel 2001, Promega Corporation 2007)