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6 votes
Accepted

Classical alignment with HISAT2

I believe the error is from featureCounts and the solution would be to add the -p flag to the ...
haci's user avatar
  • 4,032
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 ...
terdon's user avatar
  • 9,826
5 votes

How to differentiate DNA fastq and RNA fastq files?

welcome to the Bioinformatics StackExchange! This is one of those cases where the best solution is to just ask your collaborators. Don't bother with anything technical from the data itself because it'...
James Hawley's user avatar
  • 1,384
5 votes

Cannot obtain alignment summary after running Bowtie2

Remove the --quiet option, and capture STDERR as well as STDOUT: ...
Timur Shtatland's user avatar
4 votes

Issue with Differential Gene expression analysis with Deseq2

This typically happens when Age is a factor rather than numeric column, so each unique row is nested with itself. Design ~BrS_BASELINE_PATTERN + Gender is fine, ...
ATpoint's user avatar
  • 1,137
4 votes

Correlation heatmap of RNA-seq clusters all samples together leading to very low no. of DEGs

This answer has two stages, Diagnostics, what analytically has gone wrong? Where to go from here. I am pretty confident about point 1. I've been in comparable situations and I know trees. Stage 2, ...
M__'s user avatar
  • 12.1k
3 votes

How to differentiate DNA fastq and RNA fastq files?

Absolutely agree with James that you should ask your collaborators, but purely as an academic exercise... assuming you have a good reference genome+annotation (e.g. human/mouse), whole exome seq ...
Chris_Rands's user avatar
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3 votes
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DE analysis tool and method for non-coding features

The post here is useful ... https://support.bioconductor.org/p/76790/ ExactTest is really cool and completely different from ...
M__'s user avatar
  • 12.1k
3 votes

is it possible to count cell in Violin plot in seurat?

This can be a good starting point: table([email protected]$the_column_that_has_the_labels_of_interest) Basically you will be counting the number of rows (...
haci's user avatar
  • 4,032
3 votes
Accepted

Why is there antisense sequence in RNAseq data

Yes, the mRNA results should include CDSs (not only, don't forget you also have UTR sequences which are non-coding so are not part of the CDS), but there is no reason why they should all be from genes ...
terdon's user avatar
  • 9,826
3 votes
Accepted

Nextflow Error: failed to read header from "-"

This is not a Nextflow error. The problem is that without an input file, samtools sort tries to read from stdin. Using a recent ...
Steve's user avatar
  • 3,069
3 votes

Discrepancy with featurecounts analysis using a forward stranded and reverse stranded protocol

This kit uses the dUTP method to preserve strandedness. That (to my knowledge) always produces a reverse library. Not sure where you have your information of a forward library from, but we used this ...
ATpoint's user avatar
  • 1,137
3 votes

issue with rna seq analysis

Several issues: There is no non-coding RNA genome. There are non-coding genes but the genome fasta files from Ensembl are always the entire genome and this is what you should align against. Probably ...
ATpoint's user avatar
  • 1,137
3 votes

Kallisto RNA-seq errors because of incompatible indices when running <kallisto quant> using downloaded mouse index

It means that the version of the tool used for indexing and your current version are different and not compatible. Either downgrade your version to the version used for indexing, or build a new index ...
ATpoint's user avatar
  • 1,137
2 votes

Inconsistent replicate numbers in RNA-seq

It will not do any harm other than a reduction in statistical power compared to more samples per group. Common analysis tools such as DESeq2, edgeR and limma will work on unequal sample size just fine....
ATpoint's user avatar
  • 1,137
2 votes
Accepted

Nextflow Pipeline: Unexpected Input "{"

Nextflow does not have an env process directive. If stringtie is not already in your $PATH, ...
Steve's user avatar
  • 3,069
2 votes

To find genes that don't change in RNA-seq, Deseq2 has altHypothesis="lessAbs". Is there a way to make limma do the same thing for proteomics?

There is no direct support for this by best knowledge. Similar questions have been asked previously in the Bioconductor support forum, for example: https://support.bioconductor.org/p/64300/#64793 They ...
ATpoint's user avatar
  • 1,137
2 votes
Accepted

RNAseq alignment: best practices for aligning to multiple isoforms?

The fact that an alignment is unique or not is relative to the genome, not its annotation. In other words, a read will be considered a multimapper (and ignored by some programs) if it can align to ...
Alexlok's user avatar
  • 374
2 votes

how to merge more than two sample in Seurat?

As of 2023 (or Seurat >4 or >3?), simply use merge and write ...
bud.dugong's user avatar
2 votes

Integrating bulk RNA-Seq data with different sequencing depths and from different sources

Per-sample coverage / count differences are expected in all differential expression analysis, and correction for that is built into DESeq2. You shouldn't need to do any additional correction with ...
gringer's user avatar
  • 13.9k
2 votes

Test for differences between groups of samples

PCA does not do any testing and has no p-values. You may state that a separation of less than m units in PC > i is not ...
Ram RS's user avatar
  • 2,279
2 votes

How can I delete these lines in my fastq file?

This is not the thing I do these days but ... gunzip -c file.fastq.gz | sed "45500973,45500974d" > new.fastq You were using ...
M__'s user avatar
  • 12.1k
2 votes

Getting VCF file that contain common SNPs from 6 VCF file using isec

You've used the right tool and gotten the right results. What you need to understand is that VCF contains variant loci as well as the genotypes for >=1 sample(s) at those loci. The intersect ...
Ram RS's user avatar
  • 2,279
2 votes

Integrate bulk RNA and ATAC-seq genes

If I am getting your question right, all you need is https://bioinfogp.cnb.csic.es/tools/venny/. I assume that your two lists corresponding to RNA-seq and ATAC-seq use the same set of gene identifiers ...
haci's user avatar
  • 4,032
2 votes
Accepted

Applying glmnet to identify predictors for subtypes

Final answer. Keep in mind I work in Python so I'm trying to translate here. The output of coef(cv.lassoModel) ... those are your genes of interest thats your ...
M__'s user avatar
  • 12.1k
2 votes
Accepted

Paired end reads with different read lengths

I find that the forward read length is 50 while the reverse reads length is 35. How is this possible? Forward cycle length was longer on SOLiD than reverse cycle length; this is common for SOLiD data....
gringer's user avatar
  • 13.9k
2 votes

Cannot obtain alignment summary after running Bowtie2

Get samtools. Then run: samtools stats file.bam and/or samtools flagstat file.bam For example outputs see here.
Maximilian Press's user avatar
2 votes

HISAT2: RNA strandedness

According to the help produced when I type hisat2 --help, the default strandedness when running HISAT2 is unstranded: ...
gringer's user avatar
  • 13.9k
2 votes

Featurecount output .txt file from bam file

The first 6 column in standard featureCounts output represent what is in the column names. All columns after than (starting at 7) represent the counts for the sample(s). If you use a single bam file ...
ATpoint's user avatar
  • 1,137
2 votes

How to normalise Bulk RNA-seq data to account for transcript length, coverage depth, library size and batch effects?

All factors you mention bar the batch effects can be addressed by some sort of normalization (I use a modified version of UQpgQ2). Batch effects can be addressed using ComBat/SVA/ComBat-seq but my ...
Ram RS's user avatar
  • 2,279

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