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 ...
8 votes
Accepted

How can I compute gene expression for a set of RNA reads?

It's unclear if your paired-end reads are actually in fasta format, I'll presume that they're in fastq instead. The easiest tool to use is salmon, which nicely deals with things like multimapping. If ...
  • 19.3k
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 ...
  • 8,225
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-...
  • 19.3k
5 votes
Accepted

STAR-long parameters for aligning RNA ONT reads to genome

I've had great results using minimap2, particularly when combined with a pre-treatment of Canu for error correction (using minimap2 for the read-to-read mapping): ...
  • 12k
5 votes

Error creating indices using STAR

Try reducing the number of threads. When multithreading the index creation (and other memory bound tasks), the memory usage increases linearly with the number of threads. If you required a 10 GiB ...
5 votes

cDNA and alignment mapping

tl;dr Spliced aligners just make many small matches along the read pair or single read to determine splice junctions and output an alignment for the whole read, as long as it matches somewhere in the ...
  • 3,091
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

Disk space error while aligning reads using STAR

STAR versions 2.6.0b and 2.6.0a are unstable, as written Issues performing variant calling with GATK you are using version 2.6.0b. You should switch to version 2.6.0c which is stable. Also avoid to ...
3 votes
Accepted

Can I run STAR without an annotation file?

I don't think you can use the --quantMode GeneCounts option with no annotations. I think the error is trying to look for an exon file generated from the annotations ...
3 votes
Accepted

Error creating indices using STAR

In addition to the other answer (to reduce the number of threads), you might also need to add a --genomeChrBinNbits argument. Your reference has more than 5000 ...
  • 3,551
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 ...
  • 281
3 votes

How can I compute gene expression for a set of RNA reads?

In addition to salmon that was already mentioned, you can also try kallisto or RSEM, which are also fairly popular/respectable and will work with a transcriptome FASTA.
  • 2,099
3 votes

Show presence of known mutation in RNA-seq data

If you only have one gene and you only need to do this once then the simplest possible workflow is to generate the aliment using STAR (optimally with the two pass method) and open the two resultant <...
  • 1,029
3 votes

Show presence of known mutation in RNA-seq data

For counting reads I use mpileup, e.g. samtools mpileup --reference hg38.fa -r Chr10:18000-45500 input.bam, which will give base-resolution coverage for a BAM file. ...
  • 12k
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
2 votes

How can I compute gene expression for a set of RNA reads?

It's not possible to compute absolute expression from RNASeq reads if they are processed in the usual way, where a sequencer produces the same number of reads regardless of the input RNA amount. At ...
  • 12k
2 votes

How to interpret contig-alignment.psa produced by velvet

It looks like you are trying to do a transcript isoform expression analysis. Velvet is a genome assembler, and really not designed for this task. The tools you are looking for are kallisto and salmon.
  • 2,116
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
2 votes

Understanding ViennaRNA RNAdistance scoring table

Ok, the answer was right here and I missed it, posting it in case anyone else will do the same mistake as me in the future. ...
  • 141
1 vote

Is loss/gain of function reflected in RNA-seq transcript counts?

Short answer Since the effects of any given mutation is so context-dependent (and deciding which effects matter will change between researchers), the process of "variant annotation" is ...
  • 1,222
1 vote

What is the meaning of split read?

For discordant mapped reads, a position of 0 for the pair of a mapped read usually means that the pair couldn't be mapped anywhere. This would be expected for fusion events because some reads could ...
  • 12k
1 vote

Does rRNA depletion protocol give higher number of mapped reads in Intronic regions?

It's not so much that you have "intronic contamination" or "genomic contamination", rather you're not selecting explicitly for full-length mature transcripts with rRNA depletion. ...
  • 19.3k
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

What pitfalls exist with running FastQC on a bam file?

I don't see why you are trying to compare two totally different measurements. When FASTQC says a read is unique, it means that no other read shares its sequence. When STAR says a read is uniquely ...
  • 1,722
1 vote
Accepted

Why is my STAR reference genome indexing aborting on my GNU/Linux server but not on my Mac OS X laptop?

A std::bad_alloc error tends to mean that you've run out of memory. It's not unusual for head nodes to be fairly limited in capacity, so presumably that's the issue....
  • 19.3k

Only top scored, non community-wiki answers of a minimum length are eligible