11
votes
Accepted
What to use to edit RNA alignments?
I would suggest use RALEE—RNALignment Editor in Emacs. It can get for you the consensus secondary structure, you can move left/right sequences and their secondary structures (you can't do it in ...
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10
votes
Why does the SARS-Cov2 genome has letter t
We sequence and therefore typically report assemblies as DNA sequences, even if they're actually RNA.
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8
votes
Accepted
How to extract RNA sequence and secondary structure restrains from a PDB file
I suggest you take a look at rna-pdb-tools we do way more than you need! :-) The tools can get you a sequence, secondary structure and much more using various ...
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7
votes
What to use to edit RNA alignments?
There's nothing really special about RNA alignments, you can use any alignment editor, including whichever one you use for protein. That said, a classic and very useful tool for this sort of thing is ...
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7
votes
Accepted
5' and 3' bias in Rna-seq data
I expect there was a sequencing problem during the last base, where some of the reagents were running low on the sequencer. This won't pose any real problem, RNAseq aligners like STAR will just soft-...
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6
votes
Basic questions about GSEA
Use the following R package for Gene Set Enrichment analysis of RNA-seq data: seqGSEA
There is another R package (fgsea) recently published called "Fast Gene Set Enrichment Analysis" by Alexey ...
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6
votes
Basic questions about GSEA
You seem to refer to the GSEA provided by the Broad institute, (there are other GSEA algorithms).
1) You can provide whatever you wish, but if you want to know if those gene sets in which side of ...
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6
votes
How to filter ribosomal RNA from scRNA-seq data
In the paper mentioned, we used the ScaleData function in Seurat to regress out the number of reads, Rn45s abundance, and percent ribosomal gene transcripts. ...
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5
votes
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Why is ribosomal RNA difficult to remove even with Poly(A) selection?
We've found ribosomal RNA to be less of a problem with sequencing that depends on polyA, which suggests the issue might be in the library preparation, rather than the selection.
Many polyA RNA ...
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5
votes
Accepted
Gene symbol list for all protein coding genes in mice
If you want all known mouse protein coding genes, you can get the list from Ensembl's BioMart.
First, select the mouse genome:
Then, in "Filters" limit to protein coding genes only:
And ...
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4
votes
How to extract RNA sequence and secondary structure restrains from a PDB file
Context
The PDB file format is a fixed-column file format designed in 1970s for storing structural models of macromolecules. The format has been around for long time, has many uses, and although it ...
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4
votes
Estimate the length of poly-A tails from randomly-primed RNAseq data
Can’t be done. If you already sequenced then I’m afraid the money is wasted (unless of course the data is good for something else).
The standard Illumina basecaller doesn’t deal well with ...
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4
votes
How to convert featureCounts to FPKM?
I have seen many posts regarding counts to RPKM and TPM.
There’s your answer then: FPKM = RPKM. It’s simply a more accurate name.
Speaking of RPKM for paired-end data is discouraged because the ...
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4
votes
Why the t-test for a specific gene shows different value compared to differential analysis?
There is no reason your t-test should reproduce edgeR. In fact, edgeR exists because t-test is inappropriate.
edgeR does the tests by pooling information from all genes, because with the low number ...
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4
votes
Accepted
Question about Co-expression analysis and finding targets for lncRNAs
You have several options to approach this with WGCNA (weighted correlation network analysis). You can run a WGCNA on the combined set, identify modules and select those lncRNAs for further follow-up ...
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4
votes
How to calculate the Maximum Ladder Distance (MLD) of ssRNA from Dot-Bracket notation?
There are different implementations for calcuating the $\rm MLD$. One possible approach is to consider $\rm MLD$ as the heighest value of all possible mountain plots, generated by shifting the ...
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3
votes
How to calculate logCPM across all samples?
If you want the values by group then subset y to contain the samples of interest and then feed that to aveLogCPM().
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3
votes
Detecting differentially expressed genes with foldchange >= 2 and FDR < 0.05
The edgeR authors recommend that you use a relatively low logFC threshold for glmTreat such as lfc=log2(1.2). A ...
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3
votes
Accepted
Detecting differentially expressed genes with foldchange >= 2 and FDR < 0.05
Fold-change >= 2 is the same as logFC (log2(fold-change)) >= 1, so your example is doing exactly what you want. logFC is generally easier to think in and work with ...
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3
votes
Strange per sequence GC content results
Although generally I recommend people to be wary about the warnings that FastQC gives (it tends to be overly paranoid), the GC content graphs here do look odd. It's good that you've had a look at it ...
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3
votes
5' and 3' bias in Rna-seq data
I know this is an old post, but if this plot is from after trimming I would suggest a different explanation: some trimming tools remove poly-A sequences from reads. If that's the case then any read ...
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3
votes
Reads mapped to exonic, intronic and intergenic regions
As @DevonRyan mentioned, it's very likely that those samples were degraded, which is good justification for excluding them from subsequent analysis.
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3
votes
Accepted
Estimate the length of poly-A tails from randomly-primed RNAseq data
Just look for polyA tracts at the end of sequences, and count them if they're larger than ~18 bases. I've done this with MinION cDNA reads by mapping the polyA adapter sequence (with an elongated ...
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3
votes
Accepted
How to filter ribosomal RNA from scRNA-seq data
The rRNA genes in that dataset are Rn45s and Rn4.5s.
BTW, you have gene counts, not transcript counts.
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3
votes
Accepted
How to compute the chance of failing to detect a gene given the detection limit of a protocol
A 90% loss can be rephrased as a 10% chance of detecting anything. So what we want to find is the probability of detecting 0 molecules, when we start with 7 and have 10% probability of success. Once ...
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3
votes
Accepted
How can I dock a protein to a nucleic acid?
I'm not sure what you meant but you can take a look at NPDock (disclaimer we wrote that tool). If you have a structure of your protein of interest, you can dock it to the structure of your DNA/RNA of ...
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3
votes
Accepted
Selection of differential expressed genes
You can usually get away with FDR < 0.1, but that's as high as you can go. This all presumes you're doing follow-up experiments of some sort, of course. I guess you could increase the FDR more, but ...
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3
votes
Accepted
Why the t-test for a specific gene shows different value compared to differential analysis?
The $log(CPM)$ of any low-moderately expressed gene will be negative. There is nothing unexpected there.
Your statistics are inappropriate for a variety of reasons. Firstly, a CPM is not a robust ...
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3
votes
Real time transcript profiles
Answer:
There are no methods that exist for investigating transcript data inside a cell without destroying the cell. Separating RNA from cells requires essentially destroying the cells and using a ...
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3
votes
Accepted
Building RNAz on MacOS
Compile with gcc or remove inline from librna/fold.c:776 and librna/fold.c:846.
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Only top scored, non community-wiki answers of a minimum length are eligible