5 votes
Accepted

What are "split reads" and "intron clusters?"

split reads - These are read that have two or more alignments to the reference from unique region of the read. In this example a 150bp read sequenced from RNA could have base 1-75 aligning to the 3' ...
  • 2,576
4 votes
Accepted

insert size pre and post trimming

It's not an unreasonable approach. rMATs is rather picky about its input, but it seems you've noticed that. rMATs can't handle trimmed reads. It also can't handle soft-clipped reads. You might as well ...
  • 19.3k
4 votes
Accepted

Detect transcript isoform abundance for a specific gene in scRNA-seq

Transcript quantification is a difficult enough problem as it is, when you add the extra difficulty of going from the low read numbers available in scRNAseq if gets even more difficult. Added to this,...
  • 3,221
4 votes
Accepted

How can I find out the code DRIMSeq uses to make dmplotProportions objects?

I was curious how they have done it as well, so here expanded @Liopis comment : ...
4 votes
Accepted

On the same strand, for the same gene, can exons be overlapping?

I'm not quite sure what you're asking, but pretty much anything can happen in terms of exons and transcripts: Two different genes encoded on different strands can share the same genomic region as an ...
  • 12k
4 votes
Accepted

Exon-exon junctions: compare experimental transcripts to reference annotation

You can use the script that comes with minimap2: ...
  • 6,023
3 votes

Detect transcript isoform abundance for a specific gene in scRNA-seq

Looking for alternative splicing in scRNA-seq is difficult, but there is at least one tool that is specifically designed for it: DISCO. The site does not have too much info, but there is a ...
  • 2,099
3 votes

Coronavirus RNA structures?

To add a more complete answer: the current coronavirus is closely related to the SARS virus that caused the outbreak in 2004, and on which much research has been done. Here is a general review of the ...
3 votes

Practical use of RNA structure

Please look up flavivirus 'double loops' as you described them previously (post for "Coronavirus RNA') and associated RNA secondary structure anomalies for dengue virus and associated vaccine (...
  • 8,005
3 votes
Accepted

Convert local alignments to spliced alignments in SAM file

Just use minimap2 in split alignment mode to realign the reads. If that is not an option, then you could try using pysam to modify the CIGAR strings. I do not recommend this, as there are many ...
  • 2,116
3 votes

How to represent trans-spliced genes in GTF?

To my knowledge there's no defined way to deal with that in GTF. GFF3 handles trans-splicing (you'll have to scroll down to "trans-spliced transcript") by giving an individual transcript multiple ...
  • 19.3k
3 votes

Spliced vs. unspliced ratios for transcripts in RNA-seq data

I don't know about tools, but i've used the following python code to calculate the ratio of reads that overlap the 5' or 3' ends of introns or that are spliced. We sum these across all introns in a ...
  • 3,221
2 votes

Spliced vs. unspliced ratios for transcripts in RNA-seq data

Take this first comment with a grain of salt, since this isn't an area I've worked in much, but: is binary classification possible? If a gene has 3 introns, and 2 are spliced out but 1 is retained, is ...
2 votes

Calculating p-value for introns which are retained (or not) between 2 conditions

Here is how we do this: First break your gene model down into non-overlapping chunks, such that any possible gene model can be built from a combination of those chunks. E.g. ...
  • 3,221
2 votes

Workflow for identification of new splicing variants from RNA-seq data?

Is there other/better way to do do this? There is almost always a better way for pretty much anything but in this case I would stick with what your colleagues are using/suggesting. Once you have a ...
  • 3,507
2 votes

Is there a computational tool or possibility to identify mRNA isoforms from the count matrix of a bulk RNA sequencing dataset?

You can back to your bam file and use cuffdiff tools it is very able to do your job follow this to liks http://cole-trapnell-lab.github.io/cufflinks/manual/ https://github.com/Jeanielmj/...
  • 131
1 vote

Is there a computational tool or possibility to identify mRNA isoforms from the count matrix of a bulk RNA sequencing dataset?

If literally all you have is the count info by gene, you can't magic up the isoform breakdown.
  • 1,722
1 vote

Calculating p-value for introns which are retained (or not) between 2 conditions

Did you look into literature? I am not an expert in this field, but a small search on google leads me to IRFinder (ref.), a tool that can assess intron retention. My advice is to see if this tool can ...
  • 3,551
1 vote

RNA velocity: competing explanations for variable ratio of spliced to unspliced transcript

In addition to Devon Ryan's data-driven answer, I've found a clue in the text. La Manno et al have not used the full Tabula Muris: the figure legend indicates that they used only terminally ...
1 vote

RNA velocity: competing explanations for variable ratio of spliced to unspliced transcript

If cells aren't in a steady state then one wouldn't expect to see the results in (e), where there are two slopes. One would instead expect to see results more like Plekha3 in panel (c), where there's ...
  • 19.3k
1 vote

Exon-exon junctions: compare experimental transcripts to reference annotation

For future googlers, Ding et al., 2017 published a review: "Comparison of Alternative Splicing Junction Detection Tools Using RNA-Seq Data". Table 1 has the list of software in this review.
  • 2,656
1 vote

Intron retention events for a condition sample with 3 replicates

All introns will have some level of retention and I guess no intron will be 100% retained. In the absence of a comparator, you will need to work out how much intron retention an intron needs to be ...
  • 3,221

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