SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
Tophat chromosome position greater than chromosome size DNAmethylome Bioinformatics 2 07-27-2016 11:39 PM
sequencing the silenced X chromosome efoss Bioinformatics 0 05-01-2013 08:33 AM
sequencing a single chromosome asling Bioinformatics 0 12-14-2011 07:30 AM
entire and partial run ptongyoo General 0 04-22-2009 06:20 PM

Reply
 
Thread Tools
Old 05-11-2017, 06:13 AM   #1
SheilaM
Junior Member
 
Location: Canada

Join Date: May 2017
Posts: 1
Default Deep sequencing one entire chromosome

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
SheilaM is offline   Reply With Quote
Old 05-11-2017, 03:51 PM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,013
Default

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.
nucacidhunter is offline   Reply With Quote
Old 05-16-2017, 12:39 PM   #3
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,212
Default

Quote:
Originally Posted by nucacidhunter View Post
I assume you have isolated the chromosome of interest and 3 ug DNA is only from that chromosome.
Wow, you are optimistic!!!

--
Phillip
pmiguel is offline   Reply With Quote
Old 05-16-2017, 12:58 PM   #4
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,212
Default

Quote:
Originally Posted by SheilaM View Post
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...

--
Phillip
pmiguel is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 12:12 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO