6

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' end of exon2 and bases 76-150 aligning to the 5' end of exon3. This would be a split read because it have two alignments (exon2 and exon3) and those alignment ...


4

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, many scRNAseq techniques (including 10X, which is what the linked dataset is) only capture the 3' end of the gene, so you can distinguish transcript with ...


4

I was curious how they have done it as well, so here expanded @Liopis comment : wget http://bioconductor.org/packages/release/bioc/src/contrib/DRIMSeq_1.6.0.tar.gz tar -zxf DRIMSeq_1.6.0.tar.gz # if you need to find it, or use grep # find DRIMSeq -name "dm_plotProportions.R" less DRIMSeq/R/dm_plotProportions.R


4

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 not trim. You'll get a lower alignment rate, but your only other choice would be to either exclude the trimmed reads or trim everything down to the same length....


4

You can use the script that comes with minimap2: # install the k8 javascript shell curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf - cp k8-0.2.4/k8-`uname -s` $HOME/bin/k8 # assuming $HOME/bin in your $PATH # acquire the latest minimap2 git clone https://github.com/lh3/minimap2.git # print novel/...


3

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 presentation about it. It uses MISO output as the input, which in turn uses SAM.


3

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 coronavirus epidemiology, life cycle etc. I haven't found yet any materials about the RNA structures in the translatable region, however the structures in the ...


3

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 (Butantan) and the yellow fever virus and its vaccine (17D). If you are aware of "double loops", we must be aware of the association of RNA secondardy structure and ...


3

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 parents (e.g., ID=some_transspliced_gene;Parent=gene1,gene2). You could use the same methodology with GTF files, but just note that it'll break most downstream ...


3

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 gene set (we actaully use this for iCLIP analysis to see if RNA binding proteins bind pre-mRNA or spliced RNA). import pysam from collections import Counter from ...


2

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 this "spliced" or "unspliced". My first impression is that an analysis would be a bit more nuanced than a binary classification. That said, I'm aware of a ...


2

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 opportunities for subtle bugs because the SAM spec is complex. You would need to: Sort the BAM on read ID so that you can efficiently retrieve reads that you want to ...


2

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. transcript 1 |>>>>>>>>>|----------|>>>>>>>>|---|>>>>| transcript 2 |>>>>>>|-------------|>>>&...


2

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/bioinformatics-workshop/wiki/Differential-Analysis-with-Cuffdiff My best


1

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


1

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 help you, before reinventing the wheel...


1

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 differentiated cells.


1

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 more variability around the the steady state line (or perhaps no steady state line at all).


1

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.


1

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 called retained. This is a human judgement, there is no automated way of deciding this. What you need to do this is some sort of PSI calculation (percent-spliced-...


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