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Old 10-05-2011, 04:09 AM   #1
Iredc27
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Location: Montevideo, Uruguay

Join Date: Oct 2011
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Wink Sample preparation

Hi to everyone, i need your help!
I am currently working in Cytogenetics in a lab located in Montevideo (Uruguay), and i am working with a mouse cell line. I am able to identify each chromosome of this cell line, based on its unique pattern of dark and light bands. I am interested in studying the sequence of the chromosome's pair 1. Through Chromosome microdissection I physically removes the complete chromosome of pair 1 from the slide. Then i procedure to make multiple copies of the isolated Chromosome using Sigma GenomePlex Single Cell Whole Amplification Kit, that work in three step 1)fragment the chromosome dna, 2)prepare a DNA library, 3)amplification of the DNA fragment
I need to sequence the whole chromosome, but in Montevideo, we don not account even with this new and incredible technology in sequencing DNA.
I was wonderng if this amplified sample of the entire chromosome (explaind before) would work if i send it to sequence with Illumina Hiseq2000 for example?

Which other things i have to take into consideration?

I really appreciate any response, criticisms or recommendations

Ire
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Old 10-05-2011, 04:38 AM   #2
ulz_peter
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Hola Ire,

That should actually work if your DNA is clean (good 260&280nm ratio), not too fragmented and you've got a decent amount of DNA after whole genome amplification. Things you should consider are: are you really interested in the sequence of the whole chromosome or do you want to enrich some specific sequences (e.g. all exons of genes on chromosome 1), which coverage do you want to yield (you need rough sequence or do you want to check for SNPs and remember that allele balance may shift through WGA.

I am actually not using the HiSeq myself, so I do not know if the Sigma WGA Kit contains additives that might be disturbing library prep...

We have our sequences done at Macrogen, they have a HiSeq and produce nice output.


Hope that helps,
Que andes bien...
Peter
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Old 10-05-2011, 05:13 AM   #3
Iredc27
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Location: Montevideo, Uruguay

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Bravo! thanks a lot Peter!
In the catalog of the kit says that the size of the DNA fragment should range from 100-1000 bp, with the mean size~400 bp, this is too fragmented?
Recently, in this year some researchers made the whole genome sequencing from this cell line, but there is not yet current information about the sequence beyond the pair 1 chromosome.

So, for that reason i think i need the whole sequencing and a high coverage
This is correct?

Thanks again!
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Old 10-05-2011, 10:24 PM   #4
ulz_peter
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a mean size of 400 bp is indeed quite fragmented. You could change to whole genome amplification methods, that produce long fragments (I use RepliG from Qiagen for that purpose). Especially for a non-targetting approach I guess you would need longer fragments as a paired-end library prep would facilitate your analysis afterwards.
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