Questions tagged [rna-seq]
Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...
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questions with no upvoted or accepted answers
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Run cuffcompare in strand-agnostic mode
Is there a way to run Cufflinks' cuffcompare in a strand-agnostic mode?
I would like to do this because I have some RNA-seq datasets derived from an unstranded run, that should be compared to a ...
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5
votes
0
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315
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Annotating splice junctions from tophat/STAR output
Is there a way to annotate the splice junctions output from tophat/STAR output?
What I mean by annotate is can I know if it was involved in an alternative splicing event say skipped exon, MXE or ...
4
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0
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43
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Salmon Pseudo count when dealing with male and female RNA-seq data
I've generated a quant seq data that I intend to use to compare male and female gene expresion, with a focus on sexual chromosome.
For my species (three-spined stickleback), it is a classic XY sex ...
4
votes
0
answers
287
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R package equivalent to RSeQC infer_experiment to get strandedness of RNA-Seq
I am currently writing an R package that includes a module to run featureCounts (gene quantification tool) from Rsubread. I wanted to be able to specify the correct strandedness option to ...
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3
votes
0
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21
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Genome-guided transcriptome reconstruction: should I filter my reference annotation by transcript_support_level or other tags?
Is there really any advantage to filtering the GTF annotation for transcriptome reconstruction (or, for example, for pseudo-alignment quantification)? Are there any downsides (i.e. reasons why I ...
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3
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32
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Can multiplexing in Sequel II SMRTcells reduce the coverage?
I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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3
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0
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72
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Patient-sample mapping in GSE72056 dataset
I want to use the single-cell data from the following expression profiling which concerns the RNA-seq of metastatic melanoma:
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72056
The data was ...
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3
votes
1
answer
319
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"perl: warning: Setting locale failed." in RepeatMasker
I'm trying to run Repeatmasker in Linux on the command line with:
...
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3
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0
answers
845
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What could cause differing counts of R1 and R2 in Paired End Sequencing (RNASEQ)
I recently finished mapping an RNAseq run using STAR2.3.0 and noticed the read1 and read2 counts are different, according to samtools flagstat.
The map% is ~80% but the R1 R2 counts are:
R1=...
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3
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0
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115
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How do I resolve disagreements in the determination of significant genes?
I want to find some good predictors (genes). I have checked the Spearman correlation of the expression of each of 23 genes with dependent variables (responders Vs non-responders and I saw only the 5 ...
- 1,801
2
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31
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Does the contrast matrix I have made address the question I am trying to answer?
I have the following design matrix:
mm_noreps.interactions <- model.matrix(~condition*TRAPed)
Both variables are factors condition has 4 levels and TRAPed has 2 ...
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2
votes
0
answers
23
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Avoiding false anticorrelations between individual genes per cell in single-cell RNAseq
When analysing a single-cell RNAseq dataset, one question I like to ask is: At a single-cell level, are these two genes correlated (expressed together) or anticorrelated (only one or the other is ...
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2
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0
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42
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What codes represent what genes?
I want to run experiments on the data used in PatternMarkers & GWCoGAPS for novel data-driven biomarkers via whole transcriptome NMF link
So far, the paper has reduced the dimension to
ammon's ...
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2
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0
answers
220
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How to adjust by multiple variables using ComBat-Seq?
I am trying to adjust my RNA data using ComBat-Seq (from sva R package) since I realised that there are 3 batches that I need ...
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2
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24
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DeconRNASeq: Extract gene names from returned mixing proportions
This question was also asked on Biostars
I am using the Bioconductor package "DeconRNASeq" to perform tissue deconvolution. Let's say I run the following code (this is from the manual):
<...
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2
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0
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293
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How to extract reads that map exclusively to a single site with 1 or zero mismatches from BAM files
I generated BAM files (sorted by coordinates) by aligning human RNA reads against the human reference genome using BWA MEM and ...
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2
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0
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68
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Pulling out genes in a scatterplot
I'm comparing gene expression among 2 different datasets (in vivo and in vitro) I have made a heatmap showing the correlation for each and then plotted the data frame to create a scatterplot. Now I ...
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2
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0
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134
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scRNA-Seq: Account for sequencing depth and gene length?
Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length.
For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
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2
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1
answer
292
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Comparing multiple treatments to multiple other treatments in edgeR for simple effects in a complex experimental design
I am working with a RNA-seq data set in maize that has a relatively complex design. There are two levels of treatment A (nitrogen fertilizer level in the field, high or low), two levels of treatment B ...
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2
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0
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40
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Grabbing all Immune related genes with databases in R
I am having trouble grabbing specific pathway info using databases in R. I have RNAseq results and I want to remove immune related genes from the current list that I have.
With a vector of gene names/...
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2
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0
answers
1k
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GO Term heatmap plot in terms of P value or fold enrichment
I'm clustering genes in terms of expression after clustering them. I'm taking out clusters and trying to find out what kind of GO terms are coming up. I came across this figure from this paper, I want ...
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2
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47
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Mutation detection using Varscan2 on RNA sequencing for estimating tumour clones with pyclone or other package
I would like to analyze my RNAseq profiles from bulk tissue samples (Paired-End, 50M reads/sample, tumour-normal pairs) with varscan2 to detect mutations.
Then I ...
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2
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426
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Reproducing GTEx transcriptome analysis
I am willing to reproduce part of the analysis from "The human transcriptome across tissues and individuals" (Melé et all, 2015).
I downloaded GTEx v6 FPKM data in txt format from GTEx portal. I want ...
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2
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0
answers
2k
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Which R package to use for differential analysis with TPM values?
I'm using hisat2, stringtie tools for the RNA-Seq analysis. After stringtie using ballgown I get FPKM and TPM values for every gene.
I have seen that edgeR, Deseq2 can be used for Counts data. I ...
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2
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0
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475
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Filter out PCA outliers automatically
I am new to bioinformatics and PCA. What I am trying to do is to remove bad cells from a dataset that was obtained with scRNA-seq for ...
- 2,538
1
vote
1
answer
64
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What statistical test to apply for DE after CibersortX deconvolution?
This question was also asked on Biostars
I am running CibersortX in high-resolution mode (which yields estimates of gene values per sample).
After that, I want to perform DE between two conditions on ...
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1
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0
answers
32
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How to use hashsolo for demultiplexing hashtags?
I am trying to use hashsolo and I want to make sure that I have done things correctly.
I did the below:
...
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1
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0
answers
18
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Generating gene signature for sample classification and survival analysis patient cohort
This is from this paper
The LSC17 score is calculated for each patient as a linear combination of GE of these 17 genes weighted by regression coefficients that were estimated from the training data as ...
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1
vote
1
answer
69
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Can I perform Expression Analysis on this 10x dataset?
10x Genomics: 20k Human PBMCs is the dataset.
Description of the dataset:
Inputs/Libraries
Human peripheral blood mononuclear cells (PBMCs) of a healthy male donor aged 30-35 were obtained by 10x ...
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1
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0
answers
44
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Benchmarking for variant identification using RNA-seq data
I am in need to benchmark the variant identification pipeline which uses RNA seq data alone without any matched-normal. I would like to know the reference dataset (and the pipeline on which the ...
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1
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0
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23
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Number of Spots in a Spatial Seurat object
I have got an integrated seurat object of 21 Spatial samples. I want to know how many spots are in all the samples together, Is there a way I can do that? Also, Please suggest any R Packages that do ...
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1
vote
0
answers
76
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Stringtie/DEprep.py gene/transcript IDs are wrongly formatted
Hi my RNAseq workflow is ending up with wrongly formatted gene IDs (and separately transcript _IDs) after a Hisat2 ->samtools sort -> stringtie -> DEprep.py workflow.
DEprep.py outputs a ...
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1
vote
0
answers
40
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GSEA. Subsetting Gene Ontology, Biological Processes, by gene set families of interest
I have run GSEA - on Gene Ontology , Biological Processes (GO and BP hereafter) - on some Mouse bulk RNA-seq data, and am currently in the process of going through the results. To do this, I have used ...
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1
vote
0
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31
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Transcriptome analysis
I am trying to assemble reads belonging to two different readlength. Is it a valid way since I am looking for common genes among the species I am assembling.
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vote
0
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68
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RNASeq NMF clustering using what kind of expression unit?
I would need some help regarding certain points in a RNASeq gene expression analysis (sorry if there are stupid questions, its my first project and i have no capable supervision). My workflow is the ...
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1
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0
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79
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How to assess quality of WGCNA module identification in blockwisemodules()
I'm exploring WGCNA for bulkRNA sequencing analysis with human subjects. I have healthy, myocarditis, and heart failure patients (which can be further broken into ischemic or nonischemic)
I have been ...
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1
vote
1
answer
217
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Tutorial for the WGCNA: changes in heatmap colours
I am trying to reproduce the results of the R tutorial of the WGCNA package.
In section I number 5, when generating the heatmap it is quite similar to the one provided by the pdf but the colours of ...
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1
vote
0
answers
74
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Questions about CreateGeneActivityMatrix function in Seurat
Seurat V3 has a function CreateGeneActivityMatrix, which can convert ATAC peaks to gene-level's matrix.
Here is the prototype:
...
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1
vote
1
answer
61
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how to create "sample file" for the qAlign() function after trimming the reads in R
I'm an absolute beginner trying to solve this question "Align the trimmed and untrimmed reads using QuasR and plot alignment statistics, did the trimming improve alignments?"
I did trim the ...
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1
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0
answers
37
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RNAseq SNP discovery: deciding upon filters and dealing with allele expression bias
I am working with non-model plant RNA samples which we have been deep sequenced and analysed using STAR aligner under default parameters.
Aim We would like to conduct SNP discovery of these samples.
...
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1
vote
0
answers
15
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Is it possible to do cell composition for scATAC-seq data without scRNA-seq data?
Background My understanding was that if I did scATAC-seq and I have some clusters of cell groups, the only way I can label it is by correlating those groups with scRNA-seq data.
My lab ordered some ...
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1
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0
answers
41
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TGCA LAML rsem data
How do I test the gene expression differences between multiple groups?
I have normalized TCGA RSEM data, this is the dataset
...
- 1,694
1
vote
0
answers
63
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How can I get a column with real gene names in my ballgown analysis?
I am doing RNAseq analysis and I am using ballgown procedure for it.
There is an option in R to calculate differentially expressed genes and use FPKM in calculating differential gene expression, which ...
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1
vote
0
answers
34
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How to simulate replicates for DGE analysis?
I am prototyping with data visualization of DGE results, and I would like to work on the analysis pipeline before the real data is available. Currently, I only have 3 samples for wild type and 1 ...
1
vote
0
answers
15
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Taiji doesn't run Pagerank
I'm trying to use Taiji to run a pagerank on our combined bulk RNAseq and ATACseq dataset.
Here's how we run it:
taiji run --config config.yml -n 10
Here's ...
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1
vote
0
answers
71
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How to create a DESeqDataSet and define experiment design before varianceStabilizingTransformation?
I have an RNAseq count matrix consisting of 2 groups (high, low) with 6 timepoints per group (T1,T2,...,T6) and 3 replicates per timepoint (rep1, rep2, rep3). So a 2-factor design with 36 samples in ...
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1
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0
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15
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Ccle miRna data units
Does anybody know what units the mirna expression data on ccle is in? I saw the pipeline in the original paper but it doesn't mention units.
Original paper: https://www.nature.com/articles/s41586-019-...
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0
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41
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Data format for pathway based clustering of samples
I came across this paper as one of the examples from this paper, this one
Figure 2. Host Protein Alterations in Infected iAT2s
...
- 1,694
1
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0
answers
26
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TCGA dataset: different accession IDs mapped to same location?
I'm currently working on TCGA miRNA dataset. After constructing a reads matrix, I'm trying to find the isoform sequence. In my data, I have the genomic location (isoform_coords). I found that entries ...
- 11
1
vote
0
answers
88
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Query on htseq count
This question has also been asked on Biostars
I am trying to run htseq-count for carrying out rna-seq analysis for solanum tuberosum and i used the following command:
...
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