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Old 10-04-2011, 03:17 PM   #1
Iredc27
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Default sequencing request

Hi! to everyone this is the first time I write in this forum.
I need your help. I would like to sequence an entire chromosome from a cell line which was isolated and amplified by Whole Genome Amplification Kit from Sigma-Aldrich.
What i need to know from my sample to request the sequencing of it with Illumina Hiseq 2000?

Thanks to everyone and sorry for my english is not so good

Ire
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Old 10-05-2011, 04:05 AM   #2
Iredc27
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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, 09:16 AM   #3
Cofactor Genomics
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Dear Ire,
This sounds like a very interesting experiment and great work on isolating a single chromosome!

One of the first questions you should define is, "what are your goals in sequencing this chromosome?" Is it for de novo assembly or resequencing? Each of these experiments have different coverage recommendations, thus the price and amount of sequencing would differ. Also, the type of read and length could vary as well, depending on your budget.

In essence the decision tree starts at de novo or resequencing, since once reads are produced, your either assembling or aligning.

I hope this makes sense and please feel free to contact us directly (off board) if you would like for us to help you with the design.

Regards,
Jon
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Old 12-14-2011, 07:22 AM   #4
asling
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hi!
We also plan to sequence a single chromosome by using Sigma GenomePlex Single Cell Whole Amplification Kit. However our purpose is not to profile the whole chromosome, but to get a specific region's sequences in the separated chromosome. 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!

Last edited by asling; 12-14-2011 at 07:35 AM.
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Old 12-14-2011, 08:19 PM   #5
krobison
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Which amplification enzymology does the kit use? You may need to upscale your coverage numbers a bit, as some WGA kits (a) produce junk not from your template and (b) you may sequence repeatedly whatever adapters the system uses. Completely removing them from the sequence can also be a bit tricky.
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Old 12-14-2011, 11:06 PM   #6
asling
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In the manul of Sigma, they only say after the fragmentation of chromosome DNA, OmniPlex Library is then PCR amplified using universal oligonucleotide primers and a limited number of cycles, it seems it has no specific requirement on PCR enzyme. what I am not sure about is that how much coverage should we sequence ? I think it should be less coverage needed for covering a certain region than profiling a whole genome.
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Old 12-15-2011, 06:58 AM   #7
krobison
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Okay, it's the kit I thought -- I've seen Illumina data for DNA from this kit & you may see a lot of the adaptor. Some of that depends on how big the fragments going into the OmniPlex are. For example, if that step generates 2Kb fragments and then in prepping your sequencing library you shear to 200bp, then offhand one would guess about 10% of the ends will be primer -- but it will be worse because those ends already exist & shouldn't require end repair (so library prep may favor them).
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Old 12-16-2011, 05:38 AM   #8
asling
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Thanks for your reminding. It is really a problem if too many primer ended DNA is prefered by library preparation, especially the final product DNA size by using this kit varies from 200bp to 1500bp, which means far more than 10% DNA fragments for sequencing have primer end. May be the only solution is to make sure the end repair process complete reacting.
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