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Old 04-14-2015, 01:05 AM   #1
xunong
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Default How to identify known lncRNA from RNA-seq data?

Hi guys,

As I have some RNA-seq data, and have already run the TopHat-cufflinks to get the transcripts.gtf file, then how do I identify the already annotated lncRNAs from the gtf file?
Besides, I've two groups of data, one is control and the other one is gene knock down group, can I perform DE analysis of lncRNAs after identified them?

I'm new to lncRNAs, do u guys have any suggestions?
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Old 04-14-2015, 08:25 AM   #2
cascoamarillo
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I'm also interested into this kind of lncRNA analysis. Check out this recent paper:
http://biorxiv.org/content/biorxiv/e...17889.full.pdf


Quote:
Originally Posted by xunong View Post
then how do I identify the already annotated lncRNAs from the gtf file?
So, are they already annotated?
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Old 04-14-2015, 06:49 PM   #3
xunong
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Well, I downloaded a bed file of known LncRNA from Noncode databasehttp://www.bioinfo.org/noncode/index.php, for my gtf file, can I just pick up ones that have overlap with this bed file according to chr start/end site?
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Old 11-18-2015, 08:55 AM   #4
kongantik
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Xunong,

I wrote a pipeline that does what you are looking for.

http://git.io/SaFh1g
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Old 04-16-2017, 02:47 AM   #5
shakilvet
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is it possible to identify lncRNA from RNA SEQ data. As the data consists of only cDNA molecules which are synthesised from mRNA
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Old 04-17-2017, 11:16 AM   #6
kongantik
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Quote:
Originally Posted by shakilvet View Post
is it possible to identify lncRNA from RNA SEQ data. As the data consists of only cDNA molecules which are synthesised from mRNA
If you do Total RNA sequencing at high depth, you will able able to identify ncRNA. If you do mRNA-Seq, then no. Illumina provides kits for Total RNA-seq for Human, Mouse and Rat, wherein you can remove rRNA and then sequence rest of the RNA.
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Old 12-11-2017, 05:27 AM   #7
bvk
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Hello,

I'm looking for detecting known and novel lncRNAs from RNA-Seq data. I have used HISAt2 for alignment and Stringtie for Transcript assembly as mentioned in this paper [https://www.nature.com/articles/nprot.2016.095]. After I have a stringtie_merged.gtf from Stringtie (Merging transcripts from all samples) I used gffcompare (same like Cuffcompare from Tuxedo protocol) to examine how the transcripts compare with the reference annotation. This gives me annotated protein coding and already known lncRNAs.

My question is can I apply your pipeline lncRNAPipe to detect novel lncRNAs?

Thank you
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Old 12-11-2017, 11:34 AM   #8
kongantik
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From what I see, gffcompare produces new class codes for known ncRNA. The pipeline is flexible, so you can manually run rest of the modules individually.

1. Make a new output directory where you want lncRNApipe output to be stored. Let's call it lncRNApipe_12_11_2017_run1 for this example.
2. Make a new directory with name exactly as cuffcompare inside lncRNApipe_12_11_2017_run1 directory.
3. Now, move all the files produced by gffcompare to lncRNApipe_12_11_2017_run1/cuffcompare. The important file lncRNApipe looks for is the *.tracking file. Make sure that basename of the files produced by gffcompare is lncRNApipe_cuffcmp. For sake of sanity, you can recreate those files with command gffcompare -o /your/complete/path/to/lncRNApipe_12_11_2017_run1/cuffcompare/lncRNApipe_cuffcmp ....... command.
4. Now run the lncRNApipe instructing it to not identify known ncRNAs and also any class codes you want to be subjected to novel lncRNA discovery. For example, by default, transcripts with class codes i, o, u, x and e are considered for lncRNA discovery. You can mention any other class codes you want or remove them (see below how to mention class codes)


If you have any installed lncRNApipe successfully, the following command should work (Do not use params.yaml here as we are running manually). Please look at all options of each of the modules to adjust per your experiment (ex: for categorize module, --min-exons -len -max-len --linc-rna-prox etc...). To get those options, do lncRNApipe --h cat, lncRNApipe --h fetch etc...

lncRNApipe --cpu 12 --run /your/complete/path/to/lncRNApipe_12_11_2017_run1 --cat-ncRNAs "--extract-pat 'i|o|u|x|e' --ignore-genePred-err -sample-names 'your_sample_name'" --fetch "-local /your/full/path/to/reference/genome/fasta/you/used/for/transcript/assembly.fa" --cpc --rna --inf --cov-inf 0 &> lncRNApipe.`date +"%m_%d_%y_%H-%M-%S"`.log

If you encounter any error, send me the log file:

lncRNApipe --send-run-report '-email [email protected] -log lncRNApipe.log -m "What error you encountered"'

Best.

Last edited by kongantik; 12-11-2017 at 11:37 AM.
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