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A. Barplots representing the number of C to T read variants for K12 in R1 and R2 after different heat/alkaline treatment times. Colors represent duplicate experiments. B. Circular barplot representing the rate of C to T read variants in all NCNNN contexts (with N = A,T C or G) for R1 (green) and R2 (orange) for DNA-seq (left) and for 3 hours heat/alkaline treatment (RIMS-seq) for K12 (center) and BL21 strains (right). C. Proportion of C to T read variants in CCWGG (red) or CCWGG (green) contexts compared to other NCNNN or CNNNN contexts for R1 and R2 in K12 and BL21. The C to T read variants in CCWGG and CCWGG motifs represent less than 2% of all variants except in K12 (R1 only) after 10 minutes, 1 and 3 hours treatments where the CCWGG motifs represent 4.1%, 22.5% and 32.6% of all C to T read variants respectively. The increase of C to T read variants in the CCWGG context is therefore specific to R1 in K12 strain. D. Visualization of the statistically significant differences in position-specific nucleotide compositions around C to T variants in R1 compared to R2 using Two Sample Logo (Crooks et al., 2004) for the K12 sample subjected to (from top to bottom) 3H, 1H, 10 min and 0 min heat alkaline treatment.
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DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...
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... increase is linear with time with a maximum of 212 fold increase of C to T read variants in R1 compared to R2 after 14 hours of heat alkaline treatment ( Figure 1D). Next, we quantified the deamination rate at all Nm5CN sequence contexts with N being A,T,C or G and show an increase of C to T variants in R1 in all contexts (Supplementary Figure 2A). Together, these results show that a measurable deamination rate can be achieved in as soon as 10 minutes of heat alkaline deamination and that deamination efficiency is similar in all sequence contexts. ...
Context 2
... estimate the non-specific damage to the DNA leading to unwanted sequencing errors, we quantified possible imbalances for other variant types (Supplementary Figure 2B). We found that G to T variants show imbalance that is likely the result of oxidative damage resulting from sonication, a common step in library preparation between RIMS-seq and DNA-seq ( Chen et al., 2017). ...
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... comparison, all datasets were down-sampled to 5 million reads corresponding to 200X coverage of the E. coli genome and instances of high confidence C to T variants (Q score > 35) on either R1 or R2 were identified. As expected, control DNA-seq experiments show comparable numbers of C to T read variants between R1 and R2, indicating true C to T variants or errors during amplification and sequencing (Figure 2A). On the other hand, the overall number of C to T read variants in R1 is progressively elevated for 10 min, 1 hour and 3 hours of heat-alkaline treatment of the E. coli K12 samples with an overall 4 fold increase after 3 hours treatment compared to no treatment; heat-alkaline treatments did not increase the rate of C to T read variants in R2 (Figure 2A). ...
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... expected, control DNA-seq experiments show comparable numbers of C to T read variants between R1 and R2, indicating true C to T variants or errors during amplification and sequencing (Figure 2A). On the other hand, the overall number of C to T read variants in R1 is progressively elevated for 10 min, 1 hour and 3 hours of heat-alkaline treatment of the E. coli K12 samples with an overall 4 fold increase after 3 hours treatment compared to no treatment; heat-alkaline treatments did not increase the rate of C to T read variants in R2 (Figure 2A). We anticipate that the elevation of the E.coli K12 C to T read variants in R1 is due to deamination of m5C. ...
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... demonstrate this, we calculated the fraction of C to T read variants in CCWGG compared to other contexts. We observed a large elevation of the C to T read variants in the CCAGG and CCTGG contexts for K12 ( Figure 2B). As expected, the C to T read variants show no elevation at CCAGG and CCTGG contexts for the E.coli BL21 strain that is missing the dcm methylase gene ( Figure 2B). ...
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... observed a large elevation of the C to T read variants in the CCAGG and CCTGG contexts for K12 ( Figure 2B). As expected, the C to T read variants show no elevation at CCAGG and CCTGG contexts for the E.coli BL21 strain that is missing the dcm methylase gene ( Figure 2B). Thus, this C to T read variant elevation is specific to the E.coli K12 strain subjected to heat-alkaline treatments, consistent with deamination detectable only on methylated sites. ...
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... 3 hours of heat-alkaline treatment, the fraction of C to T read variants in a CCWGG context increased, rising from only 1.9 % in regular DNA-seq to ~25% of all the C to T variants. This increase is only observable in R1 of the K12 strain ( Figure 2C). Conversely, no increase can be observed in CCWGG context for which the C to T variant rate at the first C is assessed ( Figure 2C). ...
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... increase is only observable in R1 of the K12 strain ( Figure 2C). Conversely, no increase can be observed in CCWGG context for which the C to T variant rate at the first C is assessed ( Figure 2C). Thus, RIMS-seq identified the second C as the one bearing the methylation, consistent with the well described dcm methylation of E.coli K12 (Palmer andMarinus, 1994) (Marinus andMorris, 1973), highlighting the ability of RIMS-seq to identify m5C methylation at base resolution within the methylated motif. ...
Context 9
... we calculated significant (p.value < 0.01) differences in position-specific nucleotide compositions around C to T variants in R1 compared to R2 using Two Sample Logo ( Crooks et al., 2004). We found a signal consistent with the dcm methylase specificity in K12 RIMS-seq samples at one and three hours of heat alkaline treatment ( Figure 2D) demonstrating that it is possible to identify methylase specificities in genomic sequence subject to as little as 1h of alkaline treatment. These results support the application of RIMS-seq for the de novo identification of methylase specificity at base resolution. ...
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DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...
