Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

Abstract

Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. Conclusions: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

No evidence for DNA N 6 -methyladenine in mammals

“…The consistent and highly unusual pattern of mCpGs/CpGs flanking reported 6mdA sites strongly suggests that much of the reported 6mdA signal in mammals is an artifact. The tendency of SMRT-seq to overestimate 6mdA content was further demonstrated in a recent study comparing MS-based and SMRT-seq-based estimates of 6mdA in the DNA of 15 prokaryotic and eukaryotic genomes (18). Although SMRT-seq and DIP-seq are routinely used to cross-validate one another, closer inspection reveals very little overlap (1 to 8%) between these tech-niques when applied to mammalian DNA (Fig.…”

“…Specificity to guanine could not be tested as even short [4 base pairs (bp)] poly-guanine sequences form strong secondary structures (guanine tetraplexes), precluding synthesis of poly-guanine oligonucleotides. Given the vanishingly low levels of 6mdA reported in mammalian DNA (4,18), even a very low affinity for the far more abundant dA would result in detectable signal when using 6mdA antibodies. Given the affinity of 6mdA antibodies for unmethylated DNA, the frequent contamination of cultured mammalian cells with 6mdA-rich bacteria and lack of appropriate controls, the results of immunodot blot determination of 6mdA abundance in mammals is rendered invalid.…”

Adenine DNA methylation, 3D genome organization and gene expression in the parasiteTrichomonas vaginalis

“…Genome-wide distribution of 6mA varies between species (27–30, 51). An evolutionary conservation for 6mA in unicellular organisms has been proposed due to the similarities in 6mA distribution pattern and function between green algae and Tetrahymena (48).…”

“…6mA abundance was quantified as previously described (28, 51). 1.5 µg of genomic DNA in 30 ml ddH2O was digested to free nucleosides using 5 U of DNA Degradase Plus (Zymo Research) in 25 μl reactions incubated for 2 hours at 37°C.…”

Recent advances in the detection of base modifications using the Nanopore sequencer

“…Minimum 25X and 250X coverage are recommended for the detection of m6A and m5C [46]. Although 6 mA in DNA is responsible for biological functions in bacteria, its function is unclear and it is very rare in metazoan [47]. It is suggested that SMRT sequencing overestimates 6 mA in DNA in which m6A is rare.…”