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Old 05-01-2012, 12:51 AM   #1
honey
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Default ncRNA and miRNA from RNA-seq

I have a dataset paired end with enough reads and coverage. I have analyzed the RNA-seq data for Differentila splicing and expression. My question is can I analyze ncRNA/ mirRNA in the same dataset? Any link or experience will be useful.

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Old 05-01-2012, 01:38 AM   #2
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Could you please be more specific on your data . Which plateform (illumina, roche,...) ? Is the library poly-A (if yes, you can not detect ncRNA) ?
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Old 05-01-2012, 05:57 AM   #3
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Default ncRNA in RNA-seq

Yes it is poly A and Illumina plateform. What is the best way of performing an RNA-seq in Illumina to find out ncRNA as well as expression?

Thanks
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Old 05-01-2012, 07:25 AM   #4
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The methods for preparing standard RNA vs. small RNA libraries are different. Poly(A)+ selection is specific for mRNAs, while small RNA protocols exploit the size difference and/or unique chemistry of the ends. The best approach is to construct libraries of each type.
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Old 05-01-2012, 09:04 AM   #5
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You will only really be able to find poly adenylated non coding RNAs and possible miRNA precursors.

If you are truly interested in longer non coding you will need Total RNA-seq libraries. For anything smaller you need to do the small RNA protocol for things under ~50 bp.

We have also found that having all three, Total RNA, mRNA and small, while more work and cost is incredibly informative.
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Old 05-01-2012, 04:41 PM   #6
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Hi, I have two relevant questions:

(1) What's the proportion of ncRNAs without poly adenylated tails?
(2) for total RNA-seq, do we need to deplete rRNAs and tRNAs?
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Old 06-23-2012, 05:15 AM   #7
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Would it be possible to identify miRNAs and other small ncRNAs in a paired-end dataset consisting of total RNA (rRNA depleted) that has not been size-selected?

mnkyboy: If analysing total RNA, mRNA and small RNA in parallel, is it possible to add standards to the three libraries to be able to compare them quantitatively? I would like to compare the abundance of small ncRNAs and of their precursors, and possibly of their targets.

Thanks for any input!
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Old 06-24-2012, 10:02 PM   #8
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Hi Xi,
I also have the question about the propotion of poly adenylated ncRNAs.

For the sequencing of total RNA, depletion of rRNA is utterly required and rRNA propotion remains considerable even after depletion.
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Old 06-24-2012, 11:54 PM   #9
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Quote:
Originally Posted by pppppj View Post
Hi Xi,
I also have the question about the propotion of poly adenylated ncRNAs.

For the sequencing of total RNA, depletion of rRNA is utterly required and rRNA propotion remains considerable even after depletion.
Thanks. Are you from biology background?
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Old 06-27-2012, 02:12 AM   #10
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Hi Xi,

I'm Xie Peng : ) And I'm dealing with total RNA data and getting to know the protocol from our collaborators who are biologists.

It happens that our lab had Prof. Malmberg from University of Gorgia, who is scaning ncRNAs in genomes, to give a lecture today. He also doesn't have an answer to the question about proportion of poly-A ncRNAs.
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Old 06-27-2012, 06:37 AM   #11
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In RNA-seq you cannot find miRNAs, you can capture precursors of miRNAs with quite low abundance comparing to protein-coding transcripts.
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Old 06-27-2012, 07:53 AM   #12
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Quote:
Originally Posted by vebaev View Post
In RNA-seq you cannot find miRNAs, you can capture precursors of miRNAs with quite low abundance comparing to protein-coding transcripts.
In many gene annotation databases, miRNA host genes are defined as ncRNAs. As miRNAs can be embedded in introns of coding RNAs, I think these corresponding pre-miRNAs can also be different splice-forms of coding transcripts. Why did you said the abundance of pre-miRNAs were low? Is it because of the polyA-tail capture?
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Old 06-27-2012, 07:56 AM   #13
Xi Wang
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Quote:
Originally Posted by pppppj View Post
Hi Xi,

I'm Xie Peng : ) And I'm dealing with total RNA data and getting to know the protocol from our collaborators who are biologists.

It happens that our lab had Prof. Malmberg from University of Gorgia, who is scaning ncRNAs in genomes, to give a lecture today. He also doesn't have an answer to the question about proportion of poly-A ncRNAs.
Hi Nice to meet you here. ncRNA is a hot topic but lots of questions to be answered. Hopefully, you and your collaborator can answer this question :-)
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Old 06-28-2012, 12:59 AM   #14
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Quote:
Originally Posted by vebaev View Post
In RNA-seq you cannot find miRNAs, you can capture precursors of miRNAs with quite low abundance comparing to protein-coding transcripts.
Thank you. I'm just curious about the relative abundance of miRNA compared with that of mRNA, in the sense of copy number. Do you have any ideas about that?
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Old 06-28-2012, 01:02 AM   #15
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Quote:
Originally Posted by Xi Wang View Post
Hi Nice to meet you here. ncRNA is a hot topic but lots of questions to be answered. Hopefully, you and your collaborator can answer this question :-)
Yes, glad to meet you, too : ) Is ncRNA your current research interest?
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Old 06-28-2012, 01:11 AM   #16
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Quote:
Originally Posted by pppppj View Post
Thank you. I'm just curious about the relative abundance of miRNA compared with that of mRNA, in the sense of copy number. Do you have any ideas about that?
I do not remember for copy number but from our plant samples from more than 80 000 assembled contigs we identified not more than 20 miR precursors which is quite bad maybe they are not so stable after all
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