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Navigating the pitfalls of mapping DNA and RNA modifications

Abstract

Chemical modifications to nucleic acids occur across the kingdoms of life and carry important regulatory information. Reliable high-resolution mapping of these modifications is the foundation of functional and mechanistic studies, and recent methodological advances based on next-generation sequencing and long-read sequencing platforms are critical to achieving this aim. However, mapping technologies may have limitations that sometimes lead to inconsistent results. Some of these limitations are technical in nature and specific to certain types of technology. Here, however, we focus on common (yet not always widely recognized) pitfalls that are shared among frequently used mapping technologies and discuss strategies to help technology developers and users mitigate their effects. Although the emphasis is primarily on DNA modifications, RNA modifications are also discussed.

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Fig. 1: DNA and RNA modification mapping methods based on next-generation sequencing and long-read sequencing technologies.
Fig. 2: Experimental pitfalls that can lead to false positive calls during modification mapping.
Fig. 3: Analytical pitfalls that can lead to false positive calls during modification mapping.
Fig. 4: Overview of pitfalls that can lead to false negatives during modification mapping.
Fig. 5: Mitigating false positive mapping calls.

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Acknowledgements

The work was funded by R01 HG011095 (G.F.) and R35 GM139655 (G.F.) from the National Institutes of Health (NIH).

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All authors researched data for the article, made substantial contributions to discussions of the content and reviewed and edited the manuscript before submission. Y.K. and G.F. wrote the article.

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Correspondence to Gang Fang.

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Glossary

Endogenous DNA and RNA modifications

DNA and RNA modifications generated during metabolic processes in living organisms, usually catalysed by specific enzymes within the cells.

Exogenous DNA and RNA modifications

DNA and RNA modifications generated by exogenous factors that originated outside the cells, such as externally modified nucleotides that are randomly incorporated during DNA replication, or events that are directly catalysed by exogenous DNA methyltransferases in vitro or in vivo.

False discovery rates

(FDRs). These mathematical concepts describe the expected ratio of the number of false positive classifications to the total number of positive classifications. In the context of mapping DNA or RNA modifications, the FDR refers to the probability of false positive calls among the detected modification events by a mapping method.

False negative

This mathematical concept describes when a test result incorrectly indicates the absence of a condition. In the context of mapping DNA or RNA modifications, it refers to the case whereby an authentic modification event of interest is classified as either unmodified or as a different type of modification.

False negative rate

This mathematical concept describes the probability of making a false negative call for a particular test. In the context of mapping DNA or RNA modifications, it refers to the proportion of false negative calls among all the authentic modifications of interest by a mapping method.

False positive

This mathematical concept describes when a test result incorrectly indicates the presence of a condition. In the context of mapping DNA or RNA modifications, it refers to the case whereby a base is called as modified even though it is not, or a different type of modification is called as the specific modification type of interest.

False positive rate

(FPR). This mathematical concept describes the probability of making false positive calls with a particular test. In the context of mapping DNA or RNA modifications, it refers to the proportion of false positive modification calls among unmodified bases (or modified bases of other types) by a mapping method.

Heteroplasmic

The presence of more than one type of organellar genome (mitochondrial DNA (mtDNA) or plastid DNA) within a cell or individual.

Inter-pulse duration ratio

(IPD ratio). The IPD ratio is the deviation of an observed IPD (the time length between emission pulses associated with base incorporation events) from the expected IPD associated with modification-free DNA with the same flanking sequence context. The IPD ratio reflects the presence of a chemical modification of a nucleotide or its neighbouring nucleotides.

Methylation motifs

Short sequence patterns (usually 2 ~ 10 bp) that are enriched for a certain type of DNA or RNA methylation event in an organism, and which are often determined by the recognition preference of DNA or RNA methyltransferases. For example, >95% of adenines at GATC sites are methylated (N6-methyladenine (6mA)) in γ-proteobacteria, and >80% of cytosines at CpG sites are methylated (5-methylcytosine (5mC)) in the human genome.

Modification quality value

The –log10 transformed p value. In single-molecule, real-time (SMRT) sequencing, the modification quality value describes the significance of the observed inter-pulse duration (IPD) deviation from the expected level (free of modification).

Multiple hypothesis testing

In statistics, the multiple testing problem occurs when a set of statistical inferences based on the observed values are considered simultaneously. The more inferences are made, the more likely erroneous inferences become.

Restriction-modification systems

Rudimentary immune systems found in bacteria and other prokaryotic organisms, which provide defence against foreign DNA. They include a restriction enzyme, which cuts specific unmethylated DNA sequences, and the methyltransferase, which protects the same DNA sequences.

Sensitivity

Also known as the recall or true positive rate, this mathematical concept describes the probability that a positive test is truly positive. In the context of mapping DNA or RNA modifications, it refers to the probability that true modification events are successfully detected as such by a mapping method.

Specificity

Also known as the true negative rate, this mathematical concept describes the probability that a negative test is truly negative, and is expressed as specificity = 1 – false positive rate (FPR). In the context of mapping DNA or RNA modifications, it refers to the probability that a modified event detected by a mapping method truly belongs to the modification type of interest.

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Kong, Y., Mead, E.A. & Fang, G. Navigating the pitfalls of mapping DNA and RNA modifications. Nat Rev Genet (2023). https://doi.org/10.1038/s41576-022-00559-5

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