318
V. Mittard-Runte et al.
(see also Section 9.3.1) is carried out using PHRED (Ewing et al. 1998), followed
by BLAST-based (Altschul et al. 1990) vector clipping (see also Section 9.3.1). To
determine the overall progress of the project, subsets of the given sequence data are
assembled. Lander-Waterman-like (Lander and Waterman 1988) statistical analysis
graphs are created, which plot the number of gaps over the number of sequencing
reads using data generated by the CAP3 and PHRAP assembly tools.
For the second sequencing and linking step, involving sequence editing and manual assembly inspection, the Consed software package (Gordon et al. 1998) is used.
To link and polish the existing contigs, a tool called Autofinish (Gordon et al. 2001)
is applied. The tool BACCardI is used to guide the linking of contigs (Bartels et al.
2005). This latter program is able to automatically generate BAC or fosmid maps.
This optimized approach for whole genome shotgun assembly guided by a bioinformatics pipelines was successfully applied to a variety of complete bacterial
genome projects such as those of the Competence Network “Genome research on
bacteria relevant for agriculture, environment and biotechnology”, which included
the genomes of the oil-degrading marine bacterium Alcanivorax borkumensis
(Schneiker et al. 2006), the plant pathogens Clavibacter michiganensis (Gartemann
et al. 2008), Xanthomonas campestris pv. vesicatoria (Thieme et al. 2005) and pv.
campestris B100 (Vorhölter et al. 2008), the plant growth promoting bacterium
Azoarcus (Krause et al. 2006) and the biotechnologically relevant 13Mbp bacterium
Sorangium cellulosum (Schneiker et al. 2007).
The high-throughput shotgun sequencing phase is now being superseded by massively parallel sequencing techniques but sequence assembly and genome finishing
remain an important issue for genome sequencing projects.
9.1.2 Next Generation of Genome Sequencing
Sanger-based sequencing technology cannot be improved indefinitely. Cloning bias
and difficulties with sequencing regions of a genome that exhibit strong secondary
structures (“hard stops”) limit the quality of the assemblies obtained with the Sanger
method. Some of the most promising new sequencing technologies are based on
massive parallelization. The emulsion PCR method for in vitro clonal amplification is used in the pyrosequencing technology published by Margulies et al. in 2005
(commercialized by 454 Life Sciences, acquired by Roche), the polony sequencing
method (Shendure et al. 2005) and the SOLiD system (developed by Agencourt, and
later acquired by Applied Biosystems). Another method called “bridge PCR” is used
in the single base extension system distributed by Illumina. The 454 GS20 sequencing platform from 454/Roche has been proven to be efficient for the sequencing
of whole bacterial genomes (Goldberg et al. 2006) as well as for environmental
samples (Edwards et al. 2006) by eliminating many of the cloning problems that
are associated with metagenomics. At present three platforms for massively parallel
DNA sequencing are in use. These are the Roche/454 GS FLX system (standard and
titanium series) offering longer reads than the GS20 machine, the Illumina/Solexa
V. Mittard-Runte et al.
(see also Section 9.3.1) is carried out using PHRED (Ewing et al. 1998), followed
by BLAST-based (Altschul et al. 1990) vector clipping (see also Section 9.3.1). To
determine the overall progress of the project, subsets of the given sequence data are
assembled. Lander-Waterman-like (Lander and Waterman 1988) statistical analysis
graphs are created, which plot the number of gaps over the number of sequencing
reads using data generated by the CAP3 and PHRAP assembly tools.
For the second sequencing and linking step, involving sequence editing and manual assembly inspection, the Consed software package (Gordon et al. 1998) is used.
To link and polish the existing contigs, a tool called Autofinish (Gordon et al. 2001)
is applied. The tool BACCardI is used to guide the linking of contigs (Bartels et al.
2005). This latter program is able to automatically generate BAC or fosmid maps.
This optimized approach for whole genome shotgun assembly guided by a bioinformatics pipelines was successfully applied to a variety of complete bacterial
genome projects such as those of the Competence Network “Genome research on
bacteria relevant for agriculture, environment and biotechnology”, which included
the genomes of the oil-degrading marine bacterium Alcanivorax borkumensis
(Schneiker et al. 2006), the plant pathogens Clavibacter michiganensis (Gartemann
et al. 2008), Xanthomonas campestris pv. vesicatoria (Thieme et al. 2005) and pv.
campestris B100 (Vorhölter et al. 2008), the plant growth promoting bacterium
Azoarcus (Krause et al. 2006) and the biotechnologically relevant 13Mbp bacterium
Sorangium cellulosum (Schneiker et al. 2007).
The high-throughput shotgun sequencing phase is now being superseded by massively parallel sequencing techniques but sequence assembly and genome finishing
remain an important issue for genome sequencing projects.
9.1.2 Next Generation of Genome Sequencing
Sanger-based sequencing technology cannot be improved indefinitely. Cloning bias
and difficulties with sequencing regions of a genome that exhibit strong secondary
structures (“hard stops”) limit the quality of the assemblies obtained with the Sanger
method. Some of the most promising new sequencing technologies are based on
massive parallelization. The emulsion PCR method for in vitro clonal amplification is used in the pyrosequencing technology published by Margulies et al. in 2005
(commercialized by 454 Life Sciences, acquired by Roche), the polony sequencing
method (Shendure et al. 2005) and the SOLiD system (developed by Agencourt, and
later acquired by Applied Biosystems). Another method called “bridge PCR” is used
in the single base extension system distributed by Illumina. The 454 GS20 sequencing platform from 454/Roche has been proven to be efficient for the sequencing
of whole bacterial genomes (Goldberg et al. 2006) as well as for environmental
samples (Edwards et al. 2006) by eliminating many of the cloning problems that
are associated with metagenomics. At present three platforms for massively parallel
DNA sequencing are in use. These are the Roche/454 GS FLX system (standard and
titanium series) offering longer reads than the GS20 machine, the Illumina/Solexa