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Hi!
We plan to sequence a single chromosome of a mammal. Our purpose is not to profile the whole chromosome, but to get a specific region's sequence in the separated chromosome.
Here is our plan:
Firstly, isolating the chromosome by flow cytometry. Secondly, using GenomePlex® Single Cell Whole Genome Amplification Kit from sigma-ALDRICH to fragment the chromosome dna,preparing a DNA library, and amplification of the DNA fragment. Then we will use these amplified DNA as a library and feed it to Hiseq2000.This kit from sigma-ALDRICH can fragment chromosome into 400~1500 bp DNA.
We now face with some difficulties:
1)Isolating chromosome. The species in our research has no reference genome avaiable,so we even don't know which chromosome our interesing region lies on. The only information we have is the upstream and downstream sequences of this region. It seems that we have to use some sequence-specific probe to mark object chromosome, and use flow cytometry to isolate this chromosome.Do you have any better solution about this?
2) How much reads should we sequence? Our purpose is only to get the sequence of a 300kb region in this chromosome. The best situation in my mind is that we denovo a set of scaffolds ,some of them contain the known sequences in upstream and downstream region of our interesting sequence, then we can define the interval region is our object sequence. Do you think it feasible?
Thanks for help!
asling
We plan to sequence a single chromosome of a mammal. Our purpose is not to profile the whole chromosome, but to get a specific region's sequence in the separated chromosome.
Here is our plan:
Firstly, isolating the chromosome by flow cytometry. Secondly, using GenomePlex® Single Cell Whole Genome Amplification Kit from sigma-ALDRICH to fragment the chromosome dna,preparing a DNA library, and amplification of the DNA fragment. Then we will use these amplified DNA as a library and feed it to Hiseq2000.This kit from sigma-ALDRICH can fragment chromosome into 400~1500 bp DNA.
We now face with some difficulties:
1)Isolating chromosome. The species in our research has no reference genome avaiable,so we even don't know which chromosome our interesing region lies on. The only information we have is the upstream and downstream sequences of this region. It seems that we have to use some sequence-specific probe to mark object chromosome, and use flow cytometry to isolate this chromosome.Do you have any better solution about this?
2) How much reads should we sequence? Our purpose is only to get the sequence of a 300kb region in this chromosome. The best situation in my mind is that we denovo a set of scaffolds ,some of them contain the known sequences in upstream and downstream region of our interesting sequence, then we can define the interval region is our object sequence. Do you think it feasible?
Thanks for help!
asling