3.3 No self-ligation products from YNNY and RNNR adapters
To prevent self-ligation NNNN adapters, adapters YNNY and RNNR were
designed. Adapters ending with YNNY and RNNR can theoretically avoided
self-ligation occurred with NNNN adapters. The methylation site
dependent adapters have four nucleotides at 5’ extrusion. Their
self-ligation between the four wobble NNNN are complementarily paired
with 256 types of combinations. When YNNY and RNNR are designed, the Y(
C or T base) and Y is not complementary and neither the R and R.
Figure 6 illustrated the results for the three adapters ending with
YNNY, RNNR, and NNN with and without T4 ligase. Self-ligated products
from NNNN adapters were observed (lane 6). No self-ligation products
were observed from the YNNY and RNNR adapters as expected.
Discussion:
The present study developed and tested three strategies to prevent the
self-ligation of adapters used in enzyme-mediated methylation sequencing
technology. The self-ligation of the two types of methylation site
independent adapters have been largely prevented, and more required
ligation products with inserts were yielded. The self-ligation of
methylation site dependent adapters were completed avoided based on
their design.
Adapters targeting the ends yielded from either methylation-sensitive or
dependent restriction endonuclease digestion were able to be
self-ligated because of their sticking termini designed for ligation
with related ends from restriction digestion. These two types of
restriction enzymes, particularly the methylation-dependent endonuclease
is essential in methylation-dependent endonuclease mediated methylation
sequencing. Strategies to prevent adapters’ self-ligation is thus
crucial in the development of this new methylation sequencing method
(Chinese patent application number 202210881697.2). This new
methylation sequencing method may employ four types of adapters, one
methylation site-dependent and three methylation site-independent
adapters. The self-ligation of methylation site-dependent adapters are
completely prevented by eliminating any possible complementary base
paring within the four wobble nucleotides in the adapters tested. For
the three types of methylation site independent adapters, adapter with
3’ extruded T has no self-ligation issue, but its efficiency in insert
coverage of less than 30% [12, 13] prompted us to develop the other
two types of adapters tested in the present study. As illustrated in
Fig. 1 and Fig. 2, adapters with CGat at its 5’ end together with the
enzyme ClaI virtually changed an reversible reaction to unidirectional
reaction of insert-adapter ligation. For the adapter with terminal TAat
string, the combination of enzymes MseI and AseI substantially decreased
adapters’ self-ligation (Fig. 3).
As illustrated in Fig. 1, an ideal strategy should have three effects:
i. to inhibit adapters’ self-ligation, ii. to promote the production of
insert-adapter ligation, and iii. to regenerate adapters from adapters’
self-ligation products. The present study demonstrated three types of
strategies in decreasing or preventing adapters’ self-ligation, with
different advantages and disadvantages. The strategy in preventing CGat
adapters’ self-ligation is overall better than the other two as it is
fulfill all the three standards as discussed.
The insert-adapter ligated products and adapters’ self-ligated product
in methylation dependent endonuclease mediated methylation sequencing
technology are hardly separated based on their sizes as they may be
predominantly between 100 and 200 base pairs[14-16]. In real
methylation sequencing library, the size difference between a large
portion of ligation products of insert and adapters and adapters’
self-ligation is about 30-34 bp, and some are as less as 17 bp. For
example, when a palindromic methylation site is cut by MspJI
((N)13mCGmCG(N)9), the insert in the library is only 30 bp. Application
of more types of adapters to decrease or eliminate self-ligated adapters
has at least four advantages. Firstly, the three types of methylation
site-independent adapters can be combined to increase the coverage of
inserts to be assayed as more inserts are ligated with adapters.
Secondly, insert-adapter ligation is preferred over insert-insert
ligation and thus coverage of insert is further increased. Thirdly, less
self-ligated adapters in the final sequencing library will minimize
sequencing noise. Fourthly, decrease self-ligated adapters will greatly
cut the cost of methylation sequencing by maximally using the sequencing
chip with most of the clusters are formed from insert-adapter products.
These advantages are precious in methylation sequencing of single cell
and circulating free DNA samples.
In conclusion, three strategies were introduced in our newly developed
methylation-dependent endonuclease mediated methylation sequencing
method. Self-ligation of the methylation site-dependent adapters could
be theoretically eliminated by design. For the two methylation
site-independent adapters, the one with methylation sensitive enzymes is
virtually abolished by forcing the reversible reaction to a
unidirectional reaction of insert-adapter ligation. The third type of
adapter with methylation unrelated endonuclease, the combination of
enzymes MseI and AseI as an example showed its usefulness in decreasing
adapters’ self-ligation. Although these three strategies are crucial for
the development of methylation-dependent endonuclease mediated
methylation sequencing, they are not equally efficient and improvement
is expected whenever new suitable endonucleases are available. These
strategies may find more applications in the development of other
enzyme-mediated genomic sequencing technologies.