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Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

ABSTRACT: BACKGROUND:Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. OBJECTIVE:Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. METHODS:We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. RESULTS:We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. CONCLUSION:The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

SUBMITTER: Grasso C

PROVIDER: S-EPMC4775062 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

Grasso Chiara C Trevisan Morena M Fiano Valentina V Tarallo Valentina V De Marco Laura L Sacerdote Carlotta C Richiardi Lorenzo L Merletti Franco F Gillio-Tos Anna A

PloS one 20160302 3

<h4>Background</h4>Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis.<h4>Objective</h4>Aim of the study was to ass ...[more]

PMID: 26934703

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Project description:Background/objectivesThe purpose of this study was to compare the differences in tooth movements when implementing the same virtual setup on the following four different software packages: ClinCheck® Pro, Ortho Analyzer®, SureSmile®, and Ortho Insight 3D®.Materials/methodsTwenty-five adult patients treated with Invisalign® at the Case School of Dental Medicine (CWRU)'s department of orthodontics were retrospectively collected. Initial stereolithography (STL) files were obtained and imported into three software packages. The teeth were moved in order to replicate the virtual setup from ClinCheck® Pro. The final outcomes were exported from each software package. ClinCheck® Pro STL files were used as the reference while STL files produced by the other software packages were used as the targets. Best fit superimpositions were performed using Geomagic® Control X. Based on the results, tooth position was adjusted in the three software packages until the virtual setups from ClinCheck® Pro were replicated. Once confirmed, the tables containing the tooth movements were compared. The number of aligners and number of attachments automatically generated from each of the software packages were also evaluated.ResultsExtrusion/intrusion (p ≤ 0.0001) and translation buccal/lingual (p ≤ 0.0004) were significantly different among the software packages. ClinCheck® Pro and SureSmile® (p ≤ 0.000), SureSmile® and Ortho Insight 3D® (p ≤ 0.014), SureSmile® and Ortho Analyzer® (p ≤ 0.009), and Ortho Insight 3D® and Ortho Analyzer® (p ≤ 0.000) generated a significantly different number of maxillary aligners. The results varied slightly for mandibular aligners, with only ClinCheck® Pro and Ortho Insight 3D® (p ≤ 0.000), SureSmile® and Ortho Insight 3D® (p ≤ 0.000), and Ortho Insight 3D® and Ortho Analyzer® (p ≤ 0.000) exhibiting a significant difference. ClinCheck® Pro and SureSmile® (p ≤ 0.000) differed significantly in the number of attachments produced.ConclusionsThere are statistically significant differences in extrusion/intrusion, translation buccal/lingual, the number of aligners, and the number of attachments when implementing the same virtual setup on different software packages. Clinicians may need to consider this when utilizing software programs for digital diagnosis and treatment planning.

Dataset Information

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

Publications

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

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