Abstract
BackgroundWith the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. It is critical though to know the accuracy of the technique used, and to establish confidence that all of the mutations were detected.ResultsIn order to evaluate the accuracy of genome resequencing using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc., we resequenced the E. coli strain W3110 Kohara using MG1655 as a reference, both of which have been completely sequenced using traditional sequencing methods. CGS detected 7 of 8 small sequence differences, one large deletion, and 9 of 12 IS element insertions present in W3110, but did not detect a large chromosomal inversion. In addition, we confirmed that CGS also detected 2 SNPs, one deletion and 7 IS element insertions that are not present in the genome sequence, which we attribute to changes that occurred after the creation of the W3110 lambda clone library. The false positive rate for SNPs was one per 244 Kb of genome sequence.ConclusionCGS is an effective way to detect multiple mutations present in one bacterium relative to another, and while highly cost-effective, is prone to certain errors. Mutations occurring in repeated sequences or in sequences with a high degree of secondary structure may go undetected. It is also critical to follow up on regions of interest in which SNPs were not called because they often indicate deletions or IS element insertions.
Highlights
With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation
The closely related and fully sequenced E. coli strains W3110 and MG1655 represent an excellent test for the accuracy of resequencing technology
The genome sequence of strain W3110 differs from MG1655 by seven single nucleotide polymorphisms (SNPs), one 2 bp insertion, 12 IS element insertions, one deletion of 6.6 kb, and an inversion of 783.1 kb [13]
Summary
With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. Genome resequencing is the determination of a genome sequence using an already established genome sequence as a reference. In hybridization-based resequencing, the reference is necessary for the generation of microarray probes and for signal normalization. In other types of resequencing the reference is used as a scaffold for the assembly of short sequence reads. The genome to be resequenced must be substantially similar to the reference; otherwise the reference looses its effectiveness. Resequencing is useful for relating phenotype to genotype and for analyzing natural variation. It has been used to study the acquisition of antibiotic resistance [1,2], the analysis of variation in pathogenic bacteria and viruses [37], and to study the experimental evolution of bacteria and yeast [8,9,10,11].
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