Deana Martin: Welcome to NEBTV.
Today, we are talking about advances in methylome analysis.
In our “Science in 60,” we will discuss the different types of DNA methylation that
exist, and then we will speak with two of our research and development scientists.
They will talk about the limitations that exist with the current methods available for
studying DNA methylation.
And then, we will introduce a novel solution that addresses some of the challenges associated
with bisulfite sequencing, and we will review the workflow associated with this product.
Deana Martin: Let's get started.
Louise Williams: The methylome describes the genome-wide modification of DNA nucleotides
by the addition of a methyl group.
These modifications occur in a wide range of organisms, from bacteria to higher eukaryotes.
Adenines and cytosines can be methylated.
Adenine methylation is not well characterized, but has been observed in bacteria, plants,
and mammalian DNA.
Louise Williams: The most common methylation modification is found on cytosines, where
DNA methyltransferase adds a methyl group to the fifth carbon, to make the 5-methylcytosine,
or 5mC.
Five mC is often considered to be the fifth base of DNA.
Louise Williams: In humans, methylated cytosines are frequently found next to guanines, and
these are known as CpGs.
There are 28 million CpGs in the human genome, and approximately 70% of these are methylated.
Louise Williams: CpG methylation is often associated with regulating gene expression
during embryonic development, and in processes such as genomic imprinting, transposon silencing,
and X-chromosome inactivation.
Changes in methylation are also associated with diseases, such as cancer.
Louise Williams: Other cytosine modifications can occur within DNA.
These can be formed by the oxidation of 5mC by the enzyme ten-eleven translocation proteins,
or the TET proteins.
Five mC is converted into 5-hydroxymethylcytosine, then 5-formylcytosine, and finally 5-carboxycytosine.
The roles of these modifications are less well-established, but, again, are thought
to be involved in gene regulation.
Deana Martin: So, I'm here today with two of our application and development scientists,
Chaithanya Ponnaluri, and Louise Williams.
Hey guys.
Louise Williams: Hey.
Deana Martin: Thanks for joining me today.
Chaithanya P: Thank you for having us.
Deana Martin: We're here today to talk about methylome analysis.
The first question that I have for you, Louise, is what is the current gold standard for methylome
analysis?
Louise Williams: The current gold standard is bisulfite sequencing.
That's been around for next-generation sequencing for about the past 10 years.
Deana Martin: So, what are the challenges associated with bisulfite sequencing?
Chaithanya P: Bisulfite sequencing basically involves the use of chemicals, so it's harsh
pH and high temperatures, which lead to damage to the DNA.
When there's damage to the DNA, it is non-random, and you have biases that are introduced because
of that.
So, you get to see high AT, low GC coverage.
Also, because of this, you don't see even distribution of the coverage in the genome,
so you are losing all the information, which is essential.
Deana Martin: Are there any other techniques available, aside from bisulfite sequencing?
Chaithanya P: Yes, currently there are multiple methods that are available, combining both
an enzymatic step, and a chemical conversion step.
It could be either a bisulfite conversion, or another chemical which modifies cytosines.
Examples could be oxidative bisulfite sequencing, also OxBS, or aTab-seq, which is Tet-assisted
bisulfite sequencing.
So these are the two that have been available for a while.
We also have something that came up recently, which is called ACE-seq, which involves use
of APOBEC enzyme, which is currently not commercially available, but it only looks at hmC reduction,
rather than both 5-hmC, 5-hydroxymethylcytosine.
Deana Martin: I see, okay.
So, the first techniques that couple enzymatic conversion with bisulfite sequencing would
face the same challenges that bisulfite sequencing faces?
Chaithanya P: Absolutely, because the DNA still experiencing the same harsh conditions.
Deana Martin: Right.
And then the enzymatic method ACE-seq, that enzymes not currently commercially available,
correct?
Chaithanya P: No, it is not available.
So, it's a home-brewed enzyme that people have used for publishing the work.
But I think that introduces variability between batches of enzymes, and things like that.
Deana Martin: Right, right.
Could you tell us a little bit about the enzymatic method that was developed here at NEB?
Louise Williams: Yeah, sure.
So, the method that we've developed here at NEB essentially has two steps to it.
I'll talk about the second step first, because it makes more sense that way.
Louise Williams: So, the second step involves the conversion of cytosines into uracils,
and this uses the APOBEC enzyme.
But, unfortunately, it also will convert 5-methylcytosines and 5-hydroxymethylcytosines as well.
If that was the case, then we wouldn't be able to differentiate between cytosines in
their modified forms.
Louise Williams: So, in order to protect those from downstream conversion, we first do an
enzymatic conversion, an oxidation, using the TET enzyme.
What that does is it converts 5-methylC and 5-hydroxymethylC into 5-carboxyC, and 5-carboxyC
is not a substrate for APOBEC, and so in that way we can keep the integrity of the sequence
and the methylation, within the genomic DNA.
Deana Martin: I see.
And this method allows you to recognize all the different types of cytosine modifications?
Louise Williams: Yes.
Well, we'll recognize 5-methylCs and 5-hydroxymethylCs.
Deana Martin: Great, okay.
So, NEB just released a new product, the NEBNext Enzymatic Methyl-seq kit.
Can you tell us a little bit about that product?
Louise Williams: Sure.
It combines the enzymatic conversion with an upstream library construction.
To do that, we use the NEBNext Ultra II library construction.
Once we've made the library, it then undergoes an enzymatic conversion, and then it is aquified,
the library would be aquified using a new enzyme from NEB, which is a polymerase called
Q5U.
Louise Williams: The advantage of using that, is that will actually amplify through uracils.
Deana Martin: So, you covered a little bit of the workflow for that product, but can
you tell me a little bit about some of the advantages of using it?
Chaithanya P: So, as Louise was mentioning, the workflow is very straightforward.
You make a library, you do the conversion, you amplify it.
It can do this by starting it as low as 10 nanograms, and you can go up to 200 nanograms.
We also have a workflow which is for 500 nanograms input, as well.
Chaithanya P: That's a big bonus, because the bisulfite sequencing, people tend not
to go lower than 100 or 50 nanogram input, so having something which works efficiently
at 10 nanograms and also at 200 nanograms, is a big plus.
Deana Martin: I see.
Chaithanya P: The whole workflow typically takes...
You start off with your genomic DNA, you end up with your library ready for sequencing
on the Illumina platform, and you have this within a day and a half.
So, you start first thing in the morning, you have the library ready for sequencing
the next day.
Louise Williams: Yes.
Deana Martin: So, how is this product going to help researchers?
Louise Williams: The whole thing about this product is that, at the end of the day, your
DNA isn't fragmented.
And because it's not fragmented, and it also accurately represents the 5-methylCs and 5-hydroxymethylCs,
it means that researchers have more options.
They have options to look at longer pieces of DNA, if they choose to.
Just for sequencing, being able to do a 2x150 instead of a 2x76 sequencing run, is huge
in terms of being able to cover more DNA, being able to get more information in a faster
amount of time, and the ability to do other experiments such as target enrichment or other
things such as long-range PCRs.
All of these things combined will open up the methylome for so many other people to
get other research areas.
Deana Martin: If you're able to get more reads with fewer sequencing runs, ultimately it's
a cost savings, right?
Louise Williams: Yep, absolutely.
Deana Martin: You need to do less runs, which is also a nice feature.
Louise Williams: So, you actually get more coverage of the genome with less reads, as
well.
That's something we haven't mentioned before, but being able to get more for less is a huge
advantage for EM-seq.
Deana Martin: Right, right.
And then, now that these technologies are available, where do you see methylome analysis
heading, in the next few years?
Chaithanya P: I think there is a lot of buzz around using methylation information as a
biomarker in cancer detection, or early detection of different disease states, like Alzheimer's
or neurological disorders, or diabetes, and things like that.
So, it's useful to have information which is credible and accurate.
Cell-free DNA is another aspect where people are getting closer in using this technology,
FFPE, which is also highly damaged DNA, but it's very well used in the pathology field,
where they have a lot of sections already available.
So, it's useful that you have technology that applies to material that is historically present,
so you can go back and do longitudinal studies based on samples that are out there.
Chaithanya P: You can also look at developmental biology, where people are studying embryology
at different stages, during the development of a fetus, and they want to look at methylation
patterns.
There's a huge swing of methylation that happens, in the early stages of fertilization, and
then followed by changes afterwards.
It's going to open up a lot of avenues for methylation research.
Deana Martin: That is so interesting.
I love the work you're doing.
Thank you so much for joining me today, I'm excited to see what you're coming up with
next.
Chaithanya P: Thank you.
Louise Williams: Thank you.
Deana Martin: Thank you.
Speaker 4: The EM-Seq workflow is a new enzyme-based method for detection of 5-methylcytosine and
5-hydroxymethylcytosine at the single-base level within DNA.
This method minimizes damage to DNA, and provides superior performance compared to bisulfite
sequencing, including more sensitive detection of 5mC and 5hmC, uniform GC coverage, greater
mapping efficiency, and detection of more CpGs with fewer sequence reads.
Speaker 4: The workflow begins with DNA that has been mechanically sheared.
This DNA is end-repaired, dA-tailed, and ligated to the EM-seq adaptor, using NEBNext Ultra
II reagents.
The DNA is now ready to move into the EM-seq conversion reactions.
Speaker 4: The EM-seq conversion reaction is a two-step process.
The first enzymatic step involves the oxidation of 5-methylcytosine to 5-carboxycytosine using
TET2 and the glycosylation of 5-hydroxymethylcytosine using the oxidation enhancer.
These reactions protect these bases from deamination in the next step.
Speaker 4: Before the second enzymatic step, the DNA is made single-stranded using either
formamide, or sodium hydroxide.
APOBEC then deaminates cytosines into uracils.
Because 5-methylcytosines and 5-hydroxymethylcytosines were protected in the first enzymatic steps,
these forms are no longer substrates for APOBEC, and are not deaminated.
Speaker 4: The last stage of EM-seq library construction is PCR.
The EM-seq libraries are amplified using NEBNext Q5U.
This is a modified form of NEBNext Q5 polymerase that can amplify uracil-containing templates,
and in EM-seq, the primers used for amplification contain unique dual indexes, and the amplified
libraries are compatible with Illumina sequencing.
For bioinformatic analysis, pipelines used for bisulfite sequencing can be used for EM-seq.