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Using AFLP to reduce template genome size
Dear Users,
I would like to ask your opinion about the following conceptual approach.
For comparison of methylation patterns in different individual plants (Taraxacum), we 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 we do not have the resources nor facilities to sequence the entire Taraxacum genome we 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 to me 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 have.
Any help, thoughts or questions are highly appreciated.
Kind regards,
Thomas van Gurp
Msc-student Biology Wageningen University
I would like to ask your opinion about the following conceptual approach.
For comparison of methylation patterns in different individual plants (Taraxacum), we 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 we do not have the resources nor facilities to sequence the entire Taraxacum genome we 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 to me 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 have.
Any help, thoughts or questions are highly appreciated.
Kind regards,
Thomas van Gurp
Msc-student Biology Wageningen University