Deep sequencing one entire chromosome

[SheilaM]

Hello, I am new to the sequencing world but my team is considering sequencing one entire chromosome at high depth, instead of whole genome sequencing.
We have maximum 3ug DNA to work with from frozen or FFPE source.
IS there a method to make a sequencing library of one entire chromosome to sequence all coding and non-coding regions at 200x?

Thank you!

Sheila

[nucacidhunter]

I assume you have isolated the chromosome of interest and 3 ug DNA is only from that chromosome. You should be able to prep shotgun Illumina library using PCR or PCR-free methods. DNA quantity may not be enough for long read platforms without amplification.

[pmiguel]

Originally Posted by nucacidhunter

I assume you have isolated the chromosome of interest and 3 ug DNA is only from that chromosome.

Wow, you are optimistic!!!

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Phillip

[pmiguel]

Originally Posted by SheilaM

Hello, I am new to the sequencing world but my team is considering sequencing one entire chromosome at high depth, instead of whole genome sequencing.
We have maximum 3ug DNA to work with from frozen or FFPE source.
IS there a method to make a sequencing library of one entire chromosome to sequence all coding and non-coding regions at 200x?

Thank you!

Sheila

I don't think this is possible unless nucacidhunter is right and you have already isolated your chromosome of interest. Last I checked, and I will admit it has been decades, isolating specific mammalian chromosomes required a great deal of skill and some very expensive equipment to do. (I'm just guessing that your samples are from a mammal -- that seems to be the default and it isn't surprising given that nearly all of us, if not all, posting here are mammals...) Even then the amount recovered would be in the single-digit ng range at best. And I can't even envision a technology that would be able to pull intact chromosomes out of FFPE samples!

Anyway, it would probably be possible to generate a chromosome-specific biotinylated oligonucleotide probe sets that could be annealed to library molecules constructed from your samples' genomic DNA -- same technology generally used to create "exon libraries".

This would only work for sequences that were actually "chromosome specific" in the genome you are studying. That is, of course, interspersed repeats spread across multiple or all chromosomes would need to be excluded if you didn't want to pull those sequence from all chromosomes.

There are companies (eg, Agilent) who would be willing to create a custom set of chromosome-specific oligos for you if off-the-shelf sets are not commercially available.

That said, you will want to run the numbers to see if it wouldn't just be cheaper to do full genome sequences on a HiSeq X Ten or the like...

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Phillip