Question: Do you use anti-sens reads for differential gene expression analysis with Stranded Paired-End Library ?
2
gravatar for ZheFrench
3.0 years ago by
ZheFrench250
France
ZheFrench250 wrote:

I have a Total RNA TrueSeq Illumina Stranded Paired-end Ribo0 Library.

I'm using Star to get a matrice of reads count.

I dont know which column count from output to take into account to make differential gene expression analysis.

As they say here

Outputs read counts per gene into ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options:

column 1: gene ID

column 2: counts for unstranded RNA-seq

column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes)

column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse)

Select the output according to the strandedness of your data.

Note, that if you have stranded data and choose one of the columns 3 or 4, the other column (4 or 3) will give you the count of antisense reads.

I launch rseqc infer_experiment.py to check library preparatin of my data.

It returns that fraction of reads were explained by the following combination 1+-,1-+,2++,2-- meaning :

read1 mapped to ‘+’ strand indicates parental gene on ‘-‘ strand

read1 mapped to ‘-‘ strand indicates parental gene on ‘+’ strand

read2 mapped to ‘+’ strand indicates parental gene on ‘+’ strand

read2 mapped to ‘-‘ strand indicates parental gene on ‘-‘ strand

From what I understood, column4 (htseq-count -s reverse) seems to be the good count regarding the library. I have selected column 4. But I was wondering what about the column 3 (which describe antisense reads).

For differential gene expression analysis, which count I need to use ?

What to do with column 3 antisense reads ? Is it a kind of artefact ? or do I have to pull column 3 and 4 together ?

Thanks for your help.

ADD COMMENTlink modified 3.0 years ago by Devon Ryan92k • written 3.0 years ago by ZheFrench250
3
gravatar for Devon Ryan
3.0 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

You want column 4. Column 3 will presumably give you funky results when you have overlapping genes. Don't add anything together, just directly use column 4.

ADD COMMENTlink written 3.0 years ago by Devon Ryan92k

I'm coming back to your answer , sorry for the delay but what about the anti-sense reads ? How are they produce ? I mean, in which step they occur in the library prep ? What is the phenomum causing their creation ? Do we have to take care of them or are they artefact ?

ADD REPLYlink written 2.8 years ago by ZheFrench250
1

Who knows, in some cases they might indicate low levels of actual transcription, in others just random artefacts.

ADD REPLYlink written 2.8 years ago by Devon Ryan92k
1

Antisense transcripts, biological noise, enhancer RNAs or artefacts from your library prep...

ADD REPLYlink written 2.8 years ago by WouterDeCoster41k
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