Limits...
Finishing genomes with limited resources: lessons from an ensemble of microbial genomes.

Nagarajan N, Cook C, Di Bonaventura M, Ge H, Richards A, Bishop-Lilly KA, DeSalle R, Read TD, Pop M - BMC Genomics (2010)

Bottom Line: While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive.Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies.These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.

View Article: PubMed Central - HTML - PubMed

Affiliation: Computational and Mathematical Biology, Genome Institute of Singapore 127726, Singapore. niranjan@umiacs.umd.edu

ABSTRACT
While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive. Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies. Correspondingly, while draft assemblies can be obtained in days, finishing can take many months and hence the time and effort can only be justified for high-priority genomes and in large sequencing centers. In this work, we revisit this issue in light of our own experience in producing finished and nearly-finished genomes for a range of microbial species in a small-lab setting. These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.

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R. prowazekii Contig Graph. Note that the comments for Figure 3 are also valid here. This graph can be resolved into a unique in silico reconstruction of the genome.
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Figure 2: R. prowazekii Contig Graph. Note that the comments for Figure 3 are also valid here. This graph can be resolved into a unique in silico reconstruction of the genome.

Mentions: R. prowazekii is a Gram negative, aerobic bacterium that is the causative agent of epidemic typhus. In order to understand the genetic basis for phenotypic variation between various laboratory strains, a strain of R. prowazekii Madrid E was sequenced using a single run of a 454 FLX instrument to more than 100× coverage. The reads were assembled using Newbler into 197 contigs with an N50 size of 450 Kbp. Further analysis of the contig adjacency information however revealed that the entire circular genome can be reconstructed into a single gap-free sequence in silico (see Methods and Figure 2). Note that the fragmentation of the genome was not an assembler issue - assemblies using Euler-SR [9] and Celera Assembler [10] were in fact slightly worse (>260 contigs).


Finishing genomes with limited resources: lessons from an ensemble of microbial genomes.

Nagarajan N, Cook C, Di Bonaventura M, Ge H, Richards A, Bishop-Lilly KA, DeSalle R, Read TD, Pop M - BMC Genomics (2010)

R. prowazekii Contig Graph. Note that the comments for Figure 3 are also valid here. This graph can be resolved into a unique in silico reconstruction of the genome.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864248&req=5

Figure 2: R. prowazekii Contig Graph. Note that the comments for Figure 3 are also valid here. This graph can be resolved into a unique in silico reconstruction of the genome.
Mentions: R. prowazekii is a Gram negative, aerobic bacterium that is the causative agent of epidemic typhus. In order to understand the genetic basis for phenotypic variation between various laboratory strains, a strain of R. prowazekii Madrid E was sequenced using a single run of a 454 FLX instrument to more than 100× coverage. The reads were assembled using Newbler into 197 contigs with an N50 size of 450 Kbp. Further analysis of the contig adjacency information however revealed that the entire circular genome can be reconstructed into a single gap-free sequence in silico (see Methods and Figure 2). Note that the fragmentation of the genome was not an assembler issue - assemblies using Euler-SR [9] and Celera Assembler [10] were in fact slightly worse (>260 contigs).

Bottom Line: While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive.Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies.These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.

View Article: PubMed Central - HTML - PubMed

Affiliation: Computational and Mathematical Biology, Genome Institute of Singapore 127726, Singapore. niranjan@umiacs.umd.edu

ABSTRACT
While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive. Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies. Correspondingly, while draft assemblies can be obtained in days, finishing can take many months and hence the time and effort can only be justified for high-priority genomes and in large sequencing centers. In this work, we revisit this issue in light of our own experience in producing finished and nearly-finished genomes for a range of microbial species in a small-lab setting. These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.

Show MeSH