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...

86 questions with no upvoted or accepted answers
Filter by
Sorted by
Tagged with
7
votes
0answers
83 views

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 ...
6
votes
0answers
92 views

Why are TPMs per 10k or 100k in many scRNA-seq studies?

I noticed that many scRNA-seq papers normalize TPMs to 10k or 100k as opposed to 1M (as the abbreviation defines them). It doesn't really matter since you are just moving the decimal point, so why ...
4
votes
0answers
374 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 ...
3
votes
0answers
58 views

PAM50 gene expression classification

I'm looking at running the PAM50 classifier on RNA-Seq from 138 breast cancer samples. However, the R package (genefu) that's useful for this does not have a ...
3
votes
0answers
103 views

Getting genes specially up or down regulated

I have 6 RNA-seq samples like this 4 patients (005, 036, 121, 013) I have 3 tumour samples and 3 cancer models (organoid) This is PCA of log transformed data by ...
3
votes
0answers
96 views

Several models identify different which genes are significant

I want to find some good predictors (genes). This is my data, log transformed RNA-seq: ...
3
votes
0answers
59 views

RNA-Seq type and and optimal fusion detection

There are several popular types of RNA-Seq library prep which are frequently used: total RNA (with and without ribosomal depletion), mRNA-Seq/poly-A, and targeted mRNA-Seq/RNA exome. What would the ...
3
votes
0answers
230 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 ...
3
votes
0answers
187 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 ...
2
votes
0answers
28 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 ...
2
votes
0answers
58 views

How to calculate module-trait relationship when trait data is in binary format?

I have a dataset of 50 breast cancer samples. These samples are classified into four subtypes Lum A, Lum B, Her2 and Basal. I have been working with lncRNAs and protein-coding genes. To identify the ...
2
votes
1answer
23 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 ...
2
votes
0answers
32 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) ...
2
votes
0answers
46 views

Patient-sample mapping in GSE72056 dataset

I want to use the single-cell data from the following expression profiling: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72056 This is also the paper published and analyzed the data https://...
2
votes
0answers
94 views

“perl: warning: Setting locale failed.” in RepeatMasker

I'm trying to run Repeatmasker in Linux on the command line with: ...
2
votes
0answers
231 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=...
2
votes
1answer
172 views

load the phenotype data for ballgown

I am trying to reproduce the work of this paper [1], and I have run StringTie successfully, but after that I have to run Ballgown but could not understand this command: ...
2
votes
0answers
320 views

Making a bed file for RSeQC

I making a bed file for RSeQC, so it can do things like compute the number of reads from exons, introns, 5"UTRs, etc. I want to use a bed file that corresponds to my GTF file, so I use gtf2bed to ...
2
votes
1answer
121 views

sva for RNA-Seq data without known phenotype

I have been working on RNA-Seq data from two different cohorts, and they show very strong batch effect (~35% variance explained by 1st component in PCA). Since I am trying to do a class discovery from ...
2
votes
0answers
627 views

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 ...
2
votes
0answers
28 views

Mutation detection using Varscan2 on RNA sequencing for estimating tumour clones with pyclone or other package

I would like to analyze my RNA seq profiles from bulk tissue samples (Paired-End, 50M reads/sample, tumour-normal pairs) with varscan2 to detect mutations. Then I would like to use those detected ...
2
votes
0answers
273 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 ...
2
votes
1answer
69 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 ...
2
votes
0answers
941 views

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 ...
2
votes
0answers
425 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 ...
1
vote
0answers
23 views

What is a good rule of thumb for the threshold of noise versus signal for RPK in RNA seq?

I have RPK values (RNA seq) and I'm wondering what is a good rule of thumb for what is considered to be noise versus what is considered to be signal? I.e what should I choose as a threshold value for ...
1
vote
0answers
18 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, ...
1
vote
1answer
60 views

Cuffmerge: EOF marker is absent. The input is probably truncated

I need to merge my all transcripts.gtf from cufflinks output follow this command line : cuffmerge -o merged_gtf_output -p 15 -s ref.fasta -g anot.gtf assembly.txt ...
1
vote
0answers
30 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 ...
1
vote
0answers
43 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 ...
1
vote
0answers
98 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 ...
1
vote
0answers
31 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 ...
1
vote
0answers
100 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 ...
1
vote
0answers
284 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 ...
1
vote
0answers
54 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 ...
1
vote
0answers
258 views

Is it advisable to remove X and Y chromosome genes in a bulk RNA-seq dataset at the level of the count matrix?

From this link remove X and Y chromosome genes in RNA-seq data using DESeq2 pipeline, I have learned that depending on context, it is perfectly valid to remove X and Y chromosomal genes in an RNA-seq ...
1
vote
0answers
70 views

How to correctly parallelise RSeQC scripts with GNU parallel?

I have a .bam, as ouput from STAR aligner, from which I need to extract some info using RSeQC while using all the computational resources available to increase ...
1
vote
0answers
493 views

Likelihood Ratio Test in DEseq2

I have a RNA seq data which I am trying to identify DEGs. Dataset contains: Untreated - 2 replicates (time point 1st day) Treated - 4 replicates (2 replicates at 3rd day & 2 replicates at 7th ...
1
vote
0answers
289 views

Error in .computeSumFactors: cells should have non-zero library sizes

While trying to run the following code in R: ...
0
votes
0answers
27 views

Which of the Transcriptome assembly method is best for identifying novel lncRNAs?

I'm working with human samples and I'm trying to identify novel lncRNAs from tumor samples of Prostate cancer. I'm using reference based transcriptome assembly with ...
0
votes
0answers
20 views

How to find siRNA off-targets from RNA-seq data?

I have an RNA-seq dataset from siRNAs targeting different components of a molecular pathway. the problem is that the siRNA ...
0
votes
1answer
61 views

Interpreting this plot from GSEA

I have RNA-seq for two groups of patients: Responders to chemotherapy (n=9) versus ...
0
votes
0answers
13 views

What is the best way to address the question of doublets and multiplets in a single cell RNA seq data set?

I have attached a histogram plot of the number of genes per cell in a single cell RNA seq data set of lung endothelial cells. I do not find a bimodal or multimodal distribution of the number of genes ...
0
votes
0answers
50 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 ...
0
votes
0answers
32 views

a proper Design Matrix for several drug treatments with both control negative and control positive

I have a dataset of RNA-seq samples for testing different drugs on the presence of another drug. One of my samples is the normal cells with no drugs (control negative) and another is the cells with ...
0
votes
0answers
20 views

particular heatmap combinning to barplot for up and down-regulated genes

I have a list of genes (regulated up and down) by Deseq2 . I'd like to generate a heatmap of a particular combination for the barplot like in this graph : which I read in this paper: https://www....
0
votes
3answers
94 views

RNAseq biological replicates not clustering in PCA plots

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster ...
0
votes
0answers
17 views

Removing Ribosomal RNA genes from Drosophila gene counts

Hi I have seen similar questions but I still need clarification on Removing ribosomal RNA genes from a gene counts matrix (Drosophila Melanogaster) Finding a fasta file with only ribosomal genes, I ...
0
votes
0answers
14 views

Detecting Transcript Isoforms with HISAT2/Stringtie RNAseq Output

I have the following Stringtie RNA-seq output files for several samples in the image below. For additional context, I have utilized as part of my RNA-seq workflow. HISAT2, samtools, and Stringtie ...
0
votes
0answers
14 views

Suggestion for a single-cell analysis: how to orchestrate a single cell analysis in order to infer the cell types?

I'm very new to scRNA-seq data so I'm sorry if I eventually posed a trifling question. I orchestrated a sc-cell experiment passing through several steps. First of all I eliminated all the genes that ...