2 Metagenome Analysis
47
et al. 1992, Kim et al. 1996, Zimmer and Verrinder 1997). For example, Bryant
et al. recently analysed a 271 kbp metagenomic BAC clone from a phototrophic mat
community for phylogenetic marker and phototrophy genes (Bryant et al. 2007).
Nevertheless, BAC cloning is up to 100–1,000 times less efficient than fosmid
cloning due to the low efficiency of electroporation as compared to in vitro packaging and transition. Moreover, the average insert size of BAC libraries is negatively
correlated with the number of BAC clones generated (Leonardo and Sedivy 1990,
Woo et al. 1994, Sheng et al. 1995, Zimmer and Verrinder 1997). For groups working on metagenomics, especially for those focusing on soil or marine sediments,
BAC cloning is still a challenge, and often the average insert size of the constructed
metagenomic BAC libraries is not much higher than those of fosmid libraries (e.g.,
Hughes et al. 1997, Beja et al. 2000, Rondon et al. 2000, MacNeil et al. 2001,
de la Torre et al. 2003, Dumont et al. 2006). In a recent publication describing
the construction of BAC libraries from soil samples, it was noted that “. . . large
insert library construction is to some degree an idiosyncratic process. . .” (Liles et al.
2008). This clearly evokes the difficulties that may be encountered, especially during BAC cloning. Nevertheless, the larger an insert is the less laborious is genome
walking, especially in a library from a highly diverse sample.
The technical aspects of the construction of large insert libraries have been
described in detail, for example by Sambrook and Russel (2001) and by Green
et al. (1997), as well as in the manuals of cloning kits. An overview is given in
Fig. 2.3. Most of the refinements published over the years will not be discussed here
due to space restrictions. However, one important step should be mentioned. This
is the size selection of DNA fragments prior to cloning, as this greatly influences
the size and quality of large insert libraries. When constructing fosmid libraries,
the inclusion of an effective size selection step prevents chimera formation resulting from the ligation of two, or more, genome fragments with one vector molecule.
Moreover, cloning efficiency may be influenced as ligation products which are too
small or too large will not be packaged into phage heads. When constructing BAC
libraries, smaller constructs will be preferentially introduced into the host cell during electroporation, and this can lead to a smaller average insert size of the resulting
library (Osoegawa et al. 1998). For BAC libraries, size selection should be performed by pulsed field gel electrophoresis (PFGE), instead of conventional agarose
gel electrophoresis. In a regular agarose gel electrophoresis with a constant electric
field, DNA molecules larger than 15–25 kbp migrate with nearly identical mobilities. In contrast, PFGE allows size-dependent separation of DNA molecules of up
to 5 Mbp in agarose gels (Sambrook and Russel 2001). Two rounds of size selection
are preferable if large amounts of DNA are separated on the gel (Osoegawa et al.
1998, Rondon et al. 2000).
2.3.5.3 Metagenomic Library Size
For microorganisms in pure culture the library size (N) needed to cover the genome
with a given probability is defined as N = [ln (1–P)]/[(ln (1–f))], with P being
the desired probability, and f being the fractional proportion of the genome in a
47
et al. 1992, Kim et al. 1996, Zimmer and Verrinder 1997). For example, Bryant
et al. recently analysed a 271 kbp metagenomic BAC clone from a phototrophic mat
community for phylogenetic marker and phototrophy genes (Bryant et al. 2007).
Nevertheless, BAC cloning is up to 100–1,000 times less efficient than fosmid
cloning due to the low efficiency of electroporation as compared to in vitro packaging and transition. Moreover, the average insert size of BAC libraries is negatively
correlated with the number of BAC clones generated (Leonardo and Sedivy 1990,
Woo et al. 1994, Sheng et al. 1995, Zimmer and Verrinder 1997). For groups working on metagenomics, especially for those focusing on soil or marine sediments,
BAC cloning is still a challenge, and often the average insert size of the constructed
metagenomic BAC libraries is not much higher than those of fosmid libraries (e.g.,
Hughes et al. 1997, Beja et al. 2000, Rondon et al. 2000, MacNeil et al. 2001,
de la Torre et al. 2003, Dumont et al. 2006). In a recent publication describing
the construction of BAC libraries from soil samples, it was noted that “. . . large
insert library construction is to some degree an idiosyncratic process. . .” (Liles et al.
2008). This clearly evokes the difficulties that may be encountered, especially during BAC cloning. Nevertheless, the larger an insert is the less laborious is genome
walking, especially in a library from a highly diverse sample.
The technical aspects of the construction of large insert libraries have been
described in detail, for example by Sambrook and Russel (2001) and by Green
et al. (1997), as well as in the manuals of cloning kits. An overview is given in
Fig. 2.3. Most of the refinements published over the years will not be discussed here
due to space restrictions. However, one important step should be mentioned. This
is the size selection of DNA fragments prior to cloning, as this greatly influences
the size and quality of large insert libraries. When constructing fosmid libraries,
the inclusion of an effective size selection step prevents chimera formation resulting from the ligation of two, or more, genome fragments with one vector molecule.
Moreover, cloning efficiency may be influenced as ligation products which are too
small or too large will not be packaged into phage heads. When constructing BAC
libraries, smaller constructs will be preferentially introduced into the host cell during electroporation, and this can lead to a smaller average insert size of the resulting
library (Osoegawa et al. 1998). For BAC libraries, size selection should be performed by pulsed field gel electrophoresis (PFGE), instead of conventional agarose
gel electrophoresis. In a regular agarose gel electrophoresis with a constant electric
field, DNA molecules larger than 15–25 kbp migrate with nearly identical mobilities. In contrast, PFGE allows size-dependent separation of DNA molecules of up
to 5 Mbp in agarose gels (Sambrook and Russel 2001). Two rounds of size selection
are preferable if large amounts of DNA are separated on the gel (Osoegawa et al.
1998, Rondon et al. 2000).
2.3.5.3 Metagenomic Library Size
For microorganisms in pure culture the library size (N) needed to cover the genome
with a given probability is defined as N = [ln (1–P)]/[(ln (1–f))], with P being
the desired probability, and f being the fractional proportion of the genome in a