- Generally sonication of DNA is performed in a buffer of atleast 130 ul (Volume of a Covaris micro-tube). After sonication DNA needs to be purified/concentrated. So, sonication always accompanies additional purification steps that lead to loss of DNA (purification by columns will lead to a minimum loss of 10% of the DNA). Additionally, one has to check the concentration of DNA and the fragments size before proceeding.
- Bisulfite conversion of the whole genome is a harsh reaction that leads to random nicks in the DNA. Thus DNA is broken down into fragments. Subjecting the whole genome to bisulfite conversion is thus doing two steps: fragmentation and bisulfite conversion. Thus, it is advantageous as it avoids sonication and loss of DNA during the purifications steps.
- Another advantage is that there is no fragmentation induced loss of DNA after ligation (as in normal Methyl-seq where bisulfite reaction is performed post ligation. This generally leads to fragmentation of ligated library that could not be amplified).
Before commenting on the applicability of post-bisulfite library preparation to RRBS, it is wise to understand the associated library preparation steps post-bisulfite conversion of whole genome. The following picture explains the methodology (adapted from zymo research webpage)
An important consideration in the above work-flow is to convert bisulfite converted ssDNA into dsDNA. This is achieved in a process akin to cDNA preparation using random oligos. Since, the fragmentation induced by bisulfite reaction is random, random oligo seems a right choice. This protocol should work fine for preparing the library for whole genome bisulfite sequencing (Methyl-seq).
To find out the suitability of this workflow for RRBS, let us revisit the basic concepts. RRBS is inteded to enrich the CpG rich regions by digesting the DNA with MspI restriction enzyme. This will result in DNA fragments ranging from as low as 40 bp to multiple kilobases in length with identical termini. However, we choose fragments in the size range of 40-480 bp that seem to be a better representation of the CpG rich regions and promoters. In the traditional RRBS protocol, we perform bisulfite conversion post adapter ligation. So, we amplify the fragments we ‘choose’ (with some loss during the bisulfite conversion due to random fragmentation of DNA).
Let us see what happens if we subject the MspI digested DNA to bisulfite reaction prior to library preparation.
- DNA is further fragmented into smaller fragments.
- This fragmentation will skew the composition of the MspI digested fragments and the desired size range of 40-480bp does not just represent CpG rich regions. This size range now contains any region of the genome.
- Termini will not be MspI recognition motifs but random nucleotides due to chemical induced fragmentation.
- Because of the random fragmentation, even sequencing data from replicates is likely to represent CpGs from different genomic loci reducing the overlap among replicates.
- The QC of the RRBS reads is assessed by the 5′ termini (CGG/TGG). Now, because of random fragmentation, terminal nucleotides are altered!
- Even when random fragmentation doesn’t happen, another issue exists during ssDNA to dsDNA conversion. Usage of random oligos is good for randomly fragmented termini. For MspI digested termini, the major chunk are identical termini which means, random oligos may not convert the DNA at the same efficiency!
Since there are multiple issues involved, I conclude (well I have not done any experiment yet) that post-bisulfite library preparation kits are not suitable for RRBS. In my view any company that claims the suitability of this kit should do the RRBS experiments with replicates before documenting and selling these kits. I am eager to know if this has worked for RRBS. I would be willing to retract this post then!