One would like to ask about the following conceptual approach:
For comparison of methylation patterns in different individual plants (Taraxacum), they would like to assess DNA methylation polymorphisms using a bisulphite sequencing approach. However, for Taraxacum no reference genome is available, only an EST library.
Since they do not have the resources nor facilities to sequence the entire Taraxacum genome they developed the following conceptual protocol:
1. Perform a restriction enzyme digestion on genomic DNA, as commonly performed in AFLP studies
2. Ligate adapters to the restriction fragments.
3. Perform a bisulphite treatment on half of the sample.
4. Perform next generation sequencing on identical (?) subsets (using primers with selective nucleotides) of both the bisulphite converted and non-converted DNA.
Theoretical advantages of this method are:
- fragments with unique length from different samples originate from homologous locations.
- No need to sequence the entire genome.
- Detailed quantitative knowledge on methylation levels acquired (given sufficient read depth) by BS-Seq of methylation levels on nucleotide level.
Some of the things which are not clear are:
- is it possible to use primers in next-gen sequencing?
- Would there be a significant cost benefit to reducing the template size for sequencing?
- Which potential drawback would this method has?
1. yes primers are commonly used to increase template in a low cycle PCR Rx prior to sequencing. Is that what you have in mind?
2. By template you mean genome size? Knowing your genome size (which can be difficult in plants) you can estimate how many fragments a particular RE will create. Multiply by desired number of samples. Divide by amount of reads generated per lane (e.g., 80 million reads). Don't forget to factor in sequence length.
3. Have you considered taking advantage of methyl sensitive REs and perhaps doing a double digest?
Hi Mohamed
For what I understand what you are trying to do is reduced representation bisulfite sequencing http://nar.oxfordjournals.org/content/33/18/5868.full.pdf+html
So, it is possible to use with NGS. It will significantly reduce the cost of the sequencing and it qill help the analysis since you do not need a template genome.
Still, you need to keep several points in mind:
When designing your adaptors and primers you will have to select either methylated adaptors or primers that recognize the bisulfite treated version of the adaptors
If you are planning to bulk several samples on your sequencing run, you should if you want it to be cost effective, then you will have to barcode your adaptors. If you barcode your adaptors then bisulfte treatment will affect your barcodes, you need to keep this in mind. I use a modification of Genotyping By Sequencing and we have modified the analysis to account for methylation, but it is not published yet
Also, why using an AFLPapproach when you can use and MSAP approach, this will give you extra methylation information and more importantly will reduce even further the fragment population to be sequence. Make sure that you try first different enzyme combinations that generate the best fragment size for the sequencing platform that you are planning to use
Finally, if you are planning to compare the native sequence to the bisulfite treated the and you are using selective primers MAKE SURE that you do not use Cyt as a selective base because if that selective base is methylated it will amplify the same in the native and in the treated but it will not if it is not methylated.
There is a lot more I could tell with time and space. So, if you need help with the design and/or the analysis of the data let me know
Some of the things which are not clear are:
- is it possible to use primers in next-gen sequencing?
- Would there be a significant cost benefit to reducing the template size for sequencing?
- Which potential drawback would this method has?
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