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NEB Employs PacBio to Study Bacterial Methylomes; Working on Optimizing Reagents for 5-mC Detection

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New England Biolabs has been using Pacific Biosciences' RS machine to sequence bacterial genomes and study bacterial methylomes, New England Biolabs' Chief Scientific Officer Rich Roberts said during a presentation at the BGI-sponsored International Conference on Genomics in the Americas held in Sacramento, Calif., earlier this month.

Additionally, Roberts said that the company is working to develop reagents for the PacBio system that improve detection of a specific form of methylation, 5-methylcytosine, which is "tricky" to detect using the current techniques on the PacBio.

Roberts said his team at New England Biolabs turned to the PacBio system to study bacterial methylomes because of a unique feature of SMRT sequencing that enables the detection of base modifications through the system's kinetics. As the polymerase incorporates nucleotides, there is a detectable pause if a base is modified. PacBio's software flags those base-modification events in the sequence data and can identify specifically N6-methyladenine, N4-methylcytosine, and C5-methylcytosine, although identifying the latter modification is still difficult and requires greater sequencing coverage.

By looking for these specific base modifications, Roberts' team can identify and study the DNA methyltransferases responsible for making the modifications — m6A-methyltransferase, m4C-methyltransferase, and m5C-methyltransferase — which have been shown to have regulatory activity in bacteria.

Previously, methods used to study methyltransferase systems were extremely labor intensive and time consuming, Roberts said. As such, prior to using PacBio sequencing, only 29 type I methyltransferases had been characterized in terms of their recognition sequence, but in the last 18 months alone, researchers have discovered 166, he said. The vast majority of the new methyltransferase systems have been characterized by researchers from the US Department of Energy's Joint Genome Institute, with groups from the J. Craig Venter Institute, New England Biolabs, the US Food and Drug Administration, and the US Department of Agriculture also contributing, Roberts said.

Currently, Roberts said that his group is collaborating with the JGI on a paper describing their results.

Aside from the new methyltransferase systems, Roberts said that an additional 173 recognition sequences have been found, but "for which we cannot yet uniquely match the gene to the recognition sequence, usually because there is more than one possibility."

While SMRT sequencing has helped in characterizing these methyltransferase systems, it is still not ideal for detecting 5-methylcytosine and the m5C-methyltransferases, Roberts said.

The signal generated from this base modification is "often not enough to distinguish clearly from the background," he said. "As a result, no one is trying to call m5C residues except in the special cases where an m5C methyltransferase gene is cloned and the only signal is from that gene," he added.

One way to work around this problem, said Roberts, is to oxidize the 5-methylcytosine to 5-hyroxymethylcytosine and then to 5-carboxylcytosine, which does generate a good signal. This can be done with the enzyme TET1, he said, "but that is not reliable." Instead, New England Biolabs is working on an alternative that Roberts said will hopefully become commercially available by the end of the year.

Roberts said understanding the bacteria methyltransferase systems could yield insight into pathogenic organisms, including how they evolve and acquire pathogenicity genes.

For instance, he said, his team used PacBio sequencing to study the epigenetics of the Escherichia coli strain responsible for an outbreak in Germany in 2011.

Comparing the outbreak strain, TY-2482, to a strain commonly grown in laboratory settings, K12, the researchers identified key differences in the strains' methyltransferase systems.

The K12 strain is "exemplified" by the methyltransferase system MG1655, which has a "number of genes that recognize foreign methylation," Roberts said. These genes were missing from the outbreak strain, so it "lost its ability to restrict incoming DNA that is methylated."

Therefore, it was able to pick up many more restriction systems and a lot more DNA, Roberts said, including the phage with the gene that encoded for shiga toxin, causing it to become pathogenic.

"The normal systems have been lost," which protected the strain from picking up methylated DNA, he said. "So you have a strain that lost an important set of protection, allowed phages to get in, and in this way became pathogenic."

Roberts said that this same phenomenon — of pathogenic strains losing their ability to restrict incoming methylated DNA — has been seen in other bacteria strains, suggesting potential implications for monitoring outbreaks.

Moving forward, Roberts said that he plans to continue studying bacterial genomes and "characterize the methyltransferases that accompany the restriction enzymes we sell," and will also "collaborate with others by analyzing genomes with a view to matching genes and recognition sequences." The long-term goal, he said, is to be able to "accurately predict the components of [restriction-modification] systems in bacterial sequences, including making good predictions of their recognition sequences."

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