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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120 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 ...
4
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1answer
109 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 ...
4
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1answer
494 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 ...
4
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272 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
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0answers
111 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 ...
3
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0answers
241 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
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0answers
16 views

Alternatives to CAP-miRSeq for microRNA processing

I am involved in a Drosophila transcriptomics project in which we have been obtaining RNAseq data for microRNAs as well as mRNAs. In initial work we used CAP-miRSeq for the latter as the Tuxedo ...
2
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0answers
93 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
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1answer
107 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
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0answers
111 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
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0answers
54 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
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1answer
213 views

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

I'm trying to run Repeatmasker in Linux on the command line with: ...
2
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0answers
512 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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477 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
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0answers
916 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
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36 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 ...
2
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0answers
385 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
118 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
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0answers
1k 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
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0answers
442 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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23 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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0answers
18 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 ...
1
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1answer
39 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 "...
1
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0answers
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 ...
1
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0answers
48 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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0answers
12 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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0answers
26 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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0answers
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 ...
1
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0answers
76 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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0answers
131 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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0answers
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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0answers
263 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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0answers
32 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
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1answer
236 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 ...
1
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0answers
90 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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0answers
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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0answers
192 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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0answers
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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0answers
125 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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0answers
373 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
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0answers
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 ...
1
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0answers
437 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 ...
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0answers
101 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
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0answers
707 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 ...
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0answers
356 views

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

While trying to run the following code in R: ...
0
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2answers
23 views

How to input data and metadata from NCBI for RNA-Seq analysis in R

I am quite new to R programming so I hope my question is a clear one and not too confusing. So, I have downloaded some .txt and .csv files containing the raw count data (I am supposed to find out ...
0
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0answers
16 views

Should I remove days_to_death = 0 before TCGA survival analysis

Some patients in TCGA data have days_to_death = 0 and days_to_follow_up = NA vice versa. Should I remove these samples before survival analysis? What does days_to_death = 0 mean? Does it mean the ...
0
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1answer
23 views

How to find an unidentified splicing variant of a protein?

I have some data indicating there might be a splicing variant of the Arabidopsis Thaliana protein I'm studying that has not been identified. Is there a database of i.e. RNA sequences (transcriptome?) ...
0
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0answers
14 views

Why use shuffled input-sequences as background of MOTIF discovery? (from CLIP-seq data using MEME-suite)

By default, STREME (MEME-suite) uses second-order shuffles of the input sequences as background. STREME highlights its usefulness for CLIP-data, which would be my origin of sequence-inputs. In this ...
0
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1answer
21 views

how can I get expression of an inserted foreign genes?

hi we have transgenic mice with human gene inserted. if we profom rnaseq for the mice, how can we get the expression value of the human gene inserted to the mice? and use this expression to compare ...