A computer simulation analysis of the accuracy of partial genome sequencing and restriction fragment analysis in estimating genetic relationships: an application to papillomavirus DNA sequences

Baozhen Qiao,Ronald M Weigel

doi:10.1186/1471-2105-5-102

Baozhen Qiao, Ronald M Weigel

Open Access

https://doi.org/10.1186/1471-2105-5-102

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Abstract

BackgroundDetermination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods – restriction fragment analysis (RFA) and partial genome DNA sequencing – were each compared to complete DNA sequencing as the definitive standard for classification.ResultsUsing the Genbank database, 16 different types or subtypes of papillomavirus were selected as study samples, because numerous complete genome sequences were available. RFA was achieved by computer-simulated digestion. The genetic similarity of samples, based on RFA, was determined from the proportion of fragments that matched in size. DNA sequences of four specific genes (E1, E6, E7, and L1), representing partial genome sequencing, were also selected for comparison to complete genome sequencing. Laboratory error was not taken into account. Evaluation of the correlation between genetic similarity matrices (Mantel's r) and comparisons of the structure of the derived dendrograms (partition metric) indicated that partial genome sequencing (for single genes) had higher agreement with complete genome sequencing, achieving a maximum Mantel's r = 0.97 and a minimum partition metric = 10. RFA had lower agreement, with a maximum Mantel's r = 0.60 and a minimum partition metric = 18.ConclusionsThis simulation indicated that for smaller genomes, such as papillomavirus, partial genome sequencing is superior to restriction fragment analysis in representing genetic relatedness among isolates. The generalizability of these results to larger genomes, as well as the impact of laboratory error, remains to be demonstrated.

Highlights

Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny
One genotyping technique used frequently as an alternative to complete genome sequencing is restriction fragment analysis (RFA), in which restriction endonuclease enzymes cleave the genome at specific sites, producing DNA fragments that are separated by size using electrophoresis [4]
The accuracy of RFA in determining the true genetic relationships can be influenced by several factors, including the number of restriction enzymes used, the specific enzymes selected for DNA digestion, and laboratory conditions [7,8,9]

Summary

Introduction

Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. Two genotyping methods – restriction fragment analysis (RFA) and partial genome DNA sequencing – were each compared to complete DNA sequencing as the definitive standard for classification. The definitive standard for assessing genetic relatedness among organisms is the complete genome sequence of nucleotide bases [3]. One genotyping technique used frequently as an alternative to complete genome sequencing is restriction fragment analysis (RFA), in which restriction endonuclease enzymes cleave the genome at specific sites, producing DNA fragments that are separated by size using electrophoresis [4]. The accuracy of RFA in determining the true genetic relationships can be influenced by several factors, including the number of restriction enzymes used, the specific enzymes selected for DNA digestion, and laboratory conditions [7,8,9]

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