Sequencing a 2cM region of the rat genome !! (??)
lsh11 at leicester.ac.uk
Mon Jan 6 13:37:22 EST 2003
we are in possession of congenic rat strains that differ with regard to their hypotensive phenotype: The (introgressed) region imparting this phenotype is about 2 - 3cM in length and at some point in the future we may need to sequence this region, in its entirety, in order to mine all genes and look for differences ( all genes are considered potential candidates at the moment ) between the two phenotypic strains and also ascertain differences in non coding (regulatory) regions that might contribute to quantitative differences in gene expression.
Accordingly, we are considering strategies to that effect and certain questions have arisen:
1. In essence, is it feasible - using a single 3700 and either GAP 4 or consed - to sequence such a region by a 'massive' and directed primer walking effort in the space of (say) 1 year ??
2. Assuming so ( a highly contentious assumption I imagine !! ) what level of redundancy would ensure that the region is on average sequenced to something like q20 in accuracy ??
3. Alternatively, would shot gun cloning the region into something like PUC and then undertaking a massive, random sequencing effort of such clones using M13 F + R primers be more feasible ??
4. In that regard, what level of coverage would be compatible with the aforementioned accuracy ??
Thankyou for any advice proffered; I realise that my questions might, in themselves, be too simplistic but I need to accomplish this feat somehow !!
Can anyone recommend insightfull literature on this subject ??
One more thing : Based on a first draught effort, I have been informed that a region of about 2 cM in the rat genome should accommodate ( on average for coding DNA ) ~ 40 genes; Does that sound right ??
Laurence S Hall
University of Leicester
Leicester LE3 9QP
Tel.: 0116 256 3040
Fax: 0116 287 5792
E-mail: LSH11 at le.ac.uk
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