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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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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6 votes
1 answer
516 views

lower mapping rates in salmon v0.13 compared to previous versions

Hi there :) Thanks for the tool! I recently updated to the new salmon (from 0.8... its been a couple years) and I noticed that my mapping percentages change dramatically between the two versions. For ...
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5 votes
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288 views

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 ...
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3 votes
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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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114 views

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 ...
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3 votes
2 answers
142 views

How to visualize genome track of gene in specific cell-lines?

I'm trying to make a plot showing genome tracks of specific genes in specific cell-lines of RNA-seq and Chip-seq data. It should look something like this I have recently seen this encode, but in ...
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3 votes
0 answers
261 views

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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2 votes
0 answers
13 views

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 votes
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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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  • 21
2 votes
0 answers
55 views

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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  • 319
2 votes
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102 views

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 votes
1 answer
176 views

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 votes
0 answers
37 views

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 votes
1 answer
249 views

"perl: warning: Setting locale failed." in RepeatMasker

I'm trying to run Repeatmasker in Linux on the command line with: ...
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2 votes
0 answers
638 views

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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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 votes
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40 views

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 votes
0 answers
406 views

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 votes
0 answers
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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 votes
0 answers
454 views

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 ...
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1 vote
0 answers
28 views

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
25 views

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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  • 21
1 vote
0 answers
33 views

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
1 answer
47 views

Low mapping reads for RNA sequence

I am new to RNA sequencing analysis. I have RNA samples for bacteria for different treatment. I did contaminant filtration, remove adapters and checked fastQC report: attached below for one sample (...
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1 vote
0 answers
14 views

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 vote
0 answers
25 views

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 ...
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1 vote
0 answers
32 views

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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  • 103
1 vote
0 answers
24 views

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 ...
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1 vote
1 answer
45 views

Assembling all transcripts for an individual gene? (using single sequence to seed the assembly)

Let's say I have a candidate gene and I believe that in an individual sample, the genome sequence differs from the reference which then interferes with alignment. Is there a way for me to do a "...
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  • 1,498
1 vote
0 answers
11 views

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
54 views

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 vote
0 answers
13 views

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 answers
29 views

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 ...
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  • 1,640
1 vote
0 answers
21 views

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 ...
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  • 11
1 vote
0 answers
81 views

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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1 vote
0 answers
153 views

Differential Gene Expression with Replicates for some of the samples

[this question has also been posted on Biostars; some additional clarification from there has been copied into this question] I've been asked to analyse a set of samples in which their control sample ...
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1 vote
0 answers
20 views

What is the best approach to classify a patient cohort by the expression (low, int, high) of a single gene of interest?

I am working with a dataset of nearly 100 patients. I performed Salmon quantification with genecodeV34 and imported the results with tximeta. I normalised the TPM salmon output with TPM (using edgeR, ...
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1 vote
0 answers
158 views

vst() from DESeq2 vs voom() from limma

I have used both to transform my leukaemia RNA-Seq data for subsequent hierarchical clustering. The result is quite different. Some subtypes of leukaemia only form a cluster (or at least sit closer) ...
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  • 105
1 vote
0 answers
336 views

How to convert Illumina IDs to Gene Names?

I am new to the area of bioinformatics, so apologies if this is too obvious of a query. I need to analyze the RNA Seq data from GSE98455. The dataset is of the following format: ...
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1 vote
0 answers
33 views

RNAseq without depletion?

I have been asked by my supervisor to conduct an RNAseq without experimentally depleting the samples. However I am quite unsure regarding how much sequencing depth per sample I would require in order ...
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1 vote
1 answer
288 views

hisat2 --rna-strandness option and downstream htseq-count analysis

I've got some doubts on the hisat2 --rna-strandness option and its output for downstream analysis. Is it expected to see a difference in alignment and counting ...
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1 vote
0 answers
105 views

include a glimma interface in a shiny app

I am trying to code a shiny app for RNA-Seq data analysis. I would like to include glimma interactive plots in it. However, in my current interface, clicking the action button ...
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1 vote
0 answers
31 views

How to detail the specific GO terms

How can I get more specific GO terms when using clusterProfiler? I got my Dotplot by: ...
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1 vote
0 answers
230 views

Should I use log2-CPM values (voom-limma) as input for my model?

We have created a model to integrate several OMICs data, but we realized that the maximum TPM values of RNA-Seq data were so big that had unexpected effects on our results. We hypothesized that this ...
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1 vote
0 answers
34 views

How to segment genome into homozygote/heterozygote blocks based on RNA-Seq data

Given a RNA-Seq data of a progeny between two inbred lines (for example an F4 plant which was derived from two inbred lines (parents) and the F1 selfed 3 times; however this is probably not crucial ...
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1 vote
0 answers
223 views

Module preservation analysis WGCNA

I have done a WGCNA (weighted gene co-expression network analysis) analysis for various brain disorders. So along with control, I have 3 different disorders such as BPD, MDD and SCZ. Now I have to ...
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  • 1,640
1 vote
1 answer
146 views

length of 'dimnames' [1686] must match that of 'dims' [3]

Please if anyone has experience with the use of the BSEQ-SC package for the deconvolution of bulk RNA sequencing data with single cell RNA sequencing data I will be very grateful for your suggestion. ...
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  • 497
1 vote
0 answers
130 views

Error using bseqsc

I will be very grateful for any hint on how to overcome the error. I wish to deconvolve my bulk RNA seq data obtained from the lungs of mice using single cell RNA seq data. For practice, I am ...
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  • 497
1 vote
0 answers
383 views

What is the formula for Mg values in TMM normalization for RNA Seq data?

I am reading through the paper "A scaling normalization method for differential expression analysis of RNA-seq data" by Mark D Robinson, Alicia Oshlack, available here. In this paper they introduce a ...
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1 vote
0 answers
58 views

How to write the subclusters in file?

I have constructed a gene co-expression network from RNA-seq data. The network file is in edge list format of memory around 1gb which was created by calculating Pearson correlation of each gene pairs ...
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