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A mostly traditional approach improves alignment of bisulfite-converted DNA.

ABSTRACT: Cytosines in genomic DNA are sometimes methylated. This affects many biological processes and diseases. The standard way of measuring methylation is to use bisulfite, which converts unmethylated cytosines to thymines, then sequence the DNA and compare it to a reference genome sequence. We describe a method for the critical step of aligning the DNA reads to the correct genomic locations. Our method builds on classic alignment techniques, including likelihood-ratio scores and spaced seeds. In a realistic benchmark, our method has a better combination of sensitivity, specificity and speed than nine other high-throughput bisulfite aligners. This study enables more accurate and rational analysis of DNA methylation. It also illustrates how to adapt general-purpose alignment methods to a special case with distorted base patterns: this should be informative for other special cases such as ancient DNA and AT-rich genomes.

SUBMITTER: Frith MC

PROVIDER: S-EPMC3401460 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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A mostly traditional approach improves alignment of bisulfite-converted DNA.

Frith Martin C MC Mori Ryota R Asai Kiyoshi K

Nucleic acids research 20120328 13

Cytosines in genomic DNA are sometimes methylated. This affects many biological processes and diseases. The standard way of measuring methylation is to use bisulfite, which converts unmethylated cytosines to thymines, then sequence the DNA and compare it to a reference genome sequence. We describe a method for the critical step of aligning the DNA reads to the correct genomic locations. Our method builds on classic alignment techniques, including likelihood-ratio scores and spaced seeds. In a re ...[more]

PMID: 22457070

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Project description:Bisulfite (BS) conversion, which includes a series of chemical reactions using bisulfite, is a prerequisite to most DNA methylation analysis methods, and thus is an essential step in the associated research process. Unfortunately, BS conversion leads to the degradation or loss of DNA, which can hinder further downstream analysis. In addition, it is well known that incomplete BS conversion is crucial, as it causes an exaggeration of the DNA methylation level, which can adversely affect the results. Therefore, there have been many attempts to measure three key features of BS conversion: BS conversion efficiency, recovery, and degradation level. In this study, a multiplex quantitative real-time PCR system named BisQuE was suggested to simultaneously analyze three important aspects of the conversion step. By adopting cytosine-free PCR primers for two differently sized multicopy regions, the short amplicon and long amplicon were obtained from both the genomic and BS-converted DNA, thus enabling the obtaining of reliable and sensitive results and the calculation of the degradation level of the conversion step. Also, probes for detecting converted/unconverted templates and C-T indicators for inducing the formula were included in this assay to quantify BS-converted DNA in order to compute the conversion efficiency and recovery. Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE. The conversion efficiency, degradation levels, as well as recovery rates of the kits were investigated. A total of 99.61-99.90% conversion efficiency was perceived for five of the kits, while the NEBNext kit showed about 94%. The lowest degradation level was shown by the NEBNext kit, whereas the other kits were quite similar. The recovery rates of the kits were found to be within the range of 18-50%. A Qubit assay was also used to compare the recovery rate of BisQuE.

Dataset Information

A mostly traditional approach improves alignment of bisulfite-converted DNA.

Publications

A mostly traditional approach improves alignment of bisulfite-converted DNA.

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