Identifying differentially expressed lncRNA's from RNA-Seq data

Question: Identifying differentially expressed lncRNA's from RNA-Seq data

18 months ago by

Biologist • 150

Biologist • 150 wrote:

I'm very new to this lncRNA things. I'm using HISAT2, STRINGTIE and BALLGOWN pipeline for differential expression analysis.

If I'm only looking for lncRNA's, I will use the lncRNA annotation from Gencode lncRNA's [ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_24/gencode.v24.long_noncoding_RNAs.gtf.gz]

First Question:

For eg: from this paper [https://www.nature.com/articles/nprot.2016.095]

Hisat2 command: Map the reads for each sample to the reference genome

hisat2 -p 8 --dta -x chrX_data/indexes/chrX_tran -1 chrX_data/samples/ERR188044_chrX_1.fastq.gz -2 chrX_data/samples/ERR188044_chrX_2.fastq.gz -S ERR188044_chrX.sam

Stringtie command: Assemble transcripts for each sample

stringtie -p 8 -G chrX_data/genes/chrX.gtf -o ERR188044_chrX.gtf –l ERR188044 ERR188044_chrX.bam

In which step I need to use the above gtf file (lncRNA annotation from Gencode lncRNA's) [ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_24/gencode.v24.long_noncoding_RNAs.gtf.gz]

In Hisat2 or Strigtie step?

Second question:

If I want to get both protein coding RNAs and lncRNA's which gtf file should I use from Gencode [http://www.gencodegenes.org/releases/current.html] ?

In this way after differential expression analysis I will be having both differentially expressed protein coding RNA's and lncRNA's. How can I filter only lncRNA's from them?

It would be very helpful if you could clear my doubts. Thank you.

lncrna rna-seq ngs • 1.2k views

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modified 18 months ago by Devon Ryan ♦ 90k • written 18 months ago by Biologist • 150

18 months ago by

Devon Ryan ♦ 90k

Freiburg, Germany

Devon Ryan ♦ 90k wrote:

Use the GTF file with hisat2 (unless you built the index with those splice sites already) and skip stringtie entirely. You're not looking for novel lncRNAs, so this doesn't benefit you. Instead, use featureCounts and then DESeq2, edgeR, or limma/voom.

Note that you end up having to modify the GTF file to get the splice sites for hisat2. This is done with hisat2_extract_splice_sites.py, which comes with hisat2.

ADD COMMENT • link modified 18 months ago • written 18 months ago by Devon Ryan ♦ 90k

No I am also looking for novel lncRnas. Just to know how the command should be given if I need to use gtf file with hisat2?

ADD REPLY • link written 18 months ago by Biologist • 150

If you're looking for novel lncRNAs then you'll need the GTF for both (unless you built the hisat index with it, in which case you only need it with stringTie).