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Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies.

Cahill MJ, Köser CU, Ross NE, Archer JA - PLoS ONE (2010)

Bottom Line: Nonetheless, there is considerable variation amongst prokaryotes.Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study.Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats.

Methodology/principal findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads.

Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

Show MeSH
A model of repeat assembly.To unambiguously assemble a repeat (black rectangle), a read must encompass the entirety of the repeat and extend, in both directions, into unique sequence. If the repeat has a length of  nt, and the adjacent unique sequence must be at least  nt, then resolution of the repeat requires that a read starts in a  window next to the repeated sequence. The likelihood of this failing to occur in an assembly of a given number of reads of a particular length, can be estimated using an approach analogous to that used to compute sequence gaps [13], [14].
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pone-0011518-g002: A model of repeat assembly.To unambiguously assemble a repeat (black rectangle), a read must encompass the entirety of the repeat and extend, in both directions, into unique sequence. If the repeat has a length of nt, and the adjacent unique sequence must be at least nt, then resolution of the repeat requires that a read starts in a window next to the repeated sequence. The likelihood of this failing to occur in an assembly of a given number of reads of a particular length, can be estimated using an approach analogous to that used to compute sequence gaps [13], [14].

Mentions: To guarantee the correct assembly of a repeated sequence, at least one read must encompass the entirety of the repeat, and extend in both directions into adjacent unique sequence. In Figure 2, the length of the reads and of the repeat are and , respectively. To assemble the repeat, the read must extend bp into the unique flanking regions. The extent to which the read must overlap the flanking sequence will depend on the particulars of the assembly. In theory, only a single nucleotide either side of the repeat would be sufficient. This is what is assumed in subsequent analyses.


Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies.

Cahill MJ, Köser CU, Ross NE, Archer JA - PLoS ONE (2010)

A model of repeat assembly.To unambiguously assemble a repeat (black rectangle), a read must encompass the entirety of the repeat and extend, in both directions, into unique sequence. If the repeat has a length of  nt, and the adjacent unique sequence must be at least  nt, then resolution of the repeat requires that a read starts in a  window next to the repeated sequence. The likelihood of this failing to occur in an assembly of a given number of reads of a particular length, can be estimated using an approach analogous to that used to compute sequence gaps [13], [14].
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902515&req=5

pone-0011518-g002: A model of repeat assembly.To unambiguously assemble a repeat (black rectangle), a read must encompass the entirety of the repeat and extend, in both directions, into unique sequence. If the repeat has a length of nt, and the adjacent unique sequence must be at least nt, then resolution of the repeat requires that a read starts in a window next to the repeated sequence. The likelihood of this failing to occur in an assembly of a given number of reads of a particular length, can be estimated using an approach analogous to that used to compute sequence gaps [13], [14].
Mentions: To guarantee the correct assembly of a repeated sequence, at least one read must encompass the entirety of the repeat, and extend in both directions into adjacent unique sequence. In Figure 2, the length of the reads and of the repeat are and , respectively. To assemble the repeat, the read must extend bp into the unique flanking regions. The extent to which the read must overlap the flanking sequence will depend on the particulars of the assembly. In theory, only a single nucleotide either side of the repeat would be sufficient. This is what is assumed in subsequent analyses.

Bottom Line: Nonetheless, there is considerable variation amongst prokaryotes.Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study.Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats.

Methodology/principal findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads.

Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

Show MeSH