Any One please provide protocol for Analysing long noncoding RNA illumina NGS data ?

Question

[gskbioinfo143]

Hi every one i need help on accurate complete protocol on analyzing long non coding RNA illumina NGS data starting from QC parameters to DE analysis.

and

which software/ tool is good for QC analysis of RNAseq data?

i have tried using fastqc tools for rnaseq data is it good, accurate or is it better to use RSeQC and RNA-SeQC tools.

Need Help

Thank you

[WouterDeCoster]

Have a look at this post describing on how to ask questions: How To Ask Good Questions On Technical And Scientific Forums

For a problem like this I would advice you to always first look up some papers from similar research and have a look at their pipeline.

[Santosh Anand]

you may also like to visit the ENCODE pipeline https://www.encodeproject.org/rna-seq/long-rnas/

Answer 1

[EagleEye]

Are those total RNA (Transcriptome) sequencing samples? If so,

try,

Tools:

1) FastQC (Check quality of sequencing).

2) Trimmomatic or Cutadapt (if necessary, uncleaned reads for adapters)

3) STAR or HISAT2 (I would personally recommend STAR)

4) SAMstat (Check quality of alignment)

5) featureCount (Check mapping quality, MAPQ using SAMstat from alignment before starting quantification)

6) DESeq2 or edgeR (Differential expression analysis)

7) GeneSCF v1.1 (command-line)/Enrichr (Web-based) [Gene ontology or pathway analysis for differentially expressed genes, this step is only applicable for protein coding genes]

Annotation:

If you are specifically looking for/analyzing long noncoding RNAs, use annotation from Gencode lncRNAs in step 3 (alignment/assembly) and step 5 (Gene/Transcript quantification)

ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/gencode.v19.long_noncoding_RNAs.gtf.gz (HG19 genome)

You can also use the updated version, if you are going to use HG38 genome in step 3

ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_24/gencode.v24.long_noncoding_RNAs.gtf.gz

[deniz]

Hi, the post is more than 3 years ago but I have a question regarding this pipeline. I want to identify differentially expresses lncRNAs and also protein-coding genes. Therefore, is it doable to use a comprehensive gene annotation file eg hg38 or gencode.v29 at step 3 (alignment/assembly) and then lncRNA annotation file at step 5 (Gene/Transcript quantification) to identify differentially expressed lncRNAs?

[c_u]

hey, so what method did you use for DE analysis for lncRNA?

[gskbioinfo143]

yes im looking for analyzing long noncoding RNAs and annotation of it. is it possible to annotate in Galaxy? Need help

Thanks

[EagleEye]

Please ask galaxy releated question on https://biostar.usegalaxy.org , you might get quick answers.

However I found this information on biostar galaxy,

https://biostar.usegalaxy.org/p/6626/