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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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9 votes
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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 ...
aechchiki's user avatar
  • 2,676
5 votes
0 answers
335 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 ...
novicebioinforesearcher's user avatar
4 votes
0 answers
49 views

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 ...
Florent Sylvestre's user avatar
4 votes
0 answers
315 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 ...
hmgeiger's user avatar
3 votes
0 answers
132 views

how to install cibersort in R, to do deconvolution using my RNA-seq Data with public single cell data

I have RNA-seq Data from lung cancer immunotherapy patients but i don't have any single cell data from my sample, so i wanted to do a deconvolution with a public single cell data. I thought of doing ...
Rita Soares's user avatar
3 votes
0 answers
25 views

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 ...
bepoli's user avatar
  • 175
3 votes
0 answers
32 views

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 ...
Caterina's user avatar
  • 307
3 votes
0 answers
81 views

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 ...
hadi's user avatar
  • 31
3 votes
1 answer
362 views

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

I'm trying to run Repeatmasker in Linux on the command line with: ...
RNAdey's user avatar
  • 31
3 votes
0 answers
992 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=...
d_kennetz's user avatar
  • 631
3 votes
1 answer
135 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 ...
Zizogolu's user avatar
  • 2,160
2 votes
0 answers
8 views

Figuring out operons from prokaryotic RNA-Seq data

I am looking at a couple of unpaired prokaryotic RNA-Seq datasets. These data have been obtained from bacteria (cultures likely axenic, not guaranteed). My aim is to figure out whether my genes of ...
Laura's user avatar
  • 957
2 votes
0 answers
329 views

GSEA vs GSVA: Pros and cons of each method

Trying to understand when GSEA is more appropriate than GSVA and vice versa. I have seen cases when running GSEA and GSVA on the same task - compare enrichment of a geneset between two groups - gives ...
Tomas Bencomo's user avatar
2 votes
1 answer
30 views

Salmon Multiple File SCRIPT giving error

Hi I am using Salmon quantitation for multiple fastq paired files using following code ...
S_Malik's user avatar
  • 61
2 votes
0 answers
54 views

How to incorporate negative controls in DESeq2

We are doing a comparison between two outcomes (positive and negative). We could not have any positive controls as we do not have any "control" data to set as baseline, either from ...
Karthik Nair's user avatar
2 votes
0 answers
24 views

Feasible to find genetic variations of two samples using RNAseq data?

I have bulk RNAseq data from two strains of mice from Jackson Lab: C57BL/6 and B6.SJL. The former expresses a Ptprc-b allele and the latter expresses a ...
geom_na's user avatar
  • 237
2 votes
0 answers
42 views

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 ...
Angus Campbell's user avatar
2 votes
0 answers
23 views

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 ...
D Greenwood's user avatar
2 votes
0 answers
43 views

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 ...
A.Dumas's user avatar
  • 497
2 votes
0 answers
474 views

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 ...
emr2's user avatar
  • 121
2 votes
0 answers
39 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): <...
LStar's user avatar
  • 21
2 votes
0 answers
382 views

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 ...
plicht's user avatar
  • 21
2 votes
0 answers
87 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 ...
mmpp's user avatar
  • 371
2 votes
0 answers
147 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 ...
Bluescreen's user avatar
2 votes
1 answer
434 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 ...
Eddie's user avatar
  • 21
2 votes
0 answers
43 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/...
James's user avatar
  • 21
2 votes
0 answers
1k 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 ...
kcm's user avatar
  • 1,804
2 votes
0 answers
54 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 ...
user9050791's user avatar
2 votes
0 answers
462 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 ...
carlesh's user avatar
  • 121
2 votes
0 answers
2k 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 ...
beginner's user avatar
  • 631
2 votes
0 answers
483 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 ...
Nikita Vlasenko's user avatar
1 vote
1 answer
28 views

PCA of bulk RNA-seq doesn't show clustering and show large variation in general

I am very new to analyzing RNAseq and I am in a group with very little experience in this regard and I am looking for some advice. My PCA after performing DESEQ2 analysis on my dataset doesn't show ...
NotmyName's user avatar
1 vote
0 answers
39 views

How much should the 5' adapter and i7 sequence differ?

In a multiome (RNA and ATAC) project I'm working on we chanced upon a strange situation. We're not sure if it poses a problem or not and would like to know if and where I can look it up if necessary. ...
Assa Yeroslaviz's user avatar
1 vote
1 answer
19 views

time*treatment series with repeated (i.e: NOT independent) sampling of replicates

I am performing the analysis of cell cultures in suspension, untreated(U) and treatment A and B, at t0, t1 and t2, 4 replicates per treatment. The experiment started with 12 cultures, 4U, 4A and 4B, ...
Alfredo Pagliuca's user avatar
1 vote
0 answers
27 views

Assessing the quality of an assembly

I am trying to run a script that assess the quality of a transcriptomic assembly, a de novo assembly using a tool called Transrate. To install the tool I followed the prompts in https://bioconda....
thole's user avatar
  • 153
1 vote
0 answers
17 views

Find most abundant transcript isoform of a gene across different tissues in long read RNA-Seq data

I want to understand which is the most abundant variant of a gene of interest across different tissues by looking at different publicly available databases (mouse/human). I have deduced from ...
user2998764's user avatar
1 vote
0 answers
22 views

I want to know what are the main differences between two R Objects constructed for analysis of microbiome data: phyloseq and TreeSummarizedExperiment

I am looking for main differences between two R Objects constructed for analysis of microbiome data: phyloseq object and TreeSummarizedExperiment object First, I will provide a little information ...
akspat's user avatar
  • 13
1 vote
0 answers
26 views

Is it normal to have low mapping to bacterial genome in a total RNA sample of plant root-colonized bacterial cells?

I had isolated the total RNA from a sample of plant roots with colonized bacteria by excising the roots (harboring the bacterial cells) and crushing them into a powder using liquid nitrogen. The RNA ...
K_081's user avatar
  • 149
1 vote
0 answers
56 views

Continuous predictor variable to identify responder genes using DESeq2

I have samples from 45 individuals at 2 time points and these individuals were given a specific diet. The BMI of these individuals were recorded at both time points. The responders to the diet are the ...
Angelo's user avatar
  • 237
1 vote
0 answers
44 views

What method uses "diverge probability" in RNA-Seq differential expression analysis?

I am replicating differential expression analysis from a paper (here) on RNA-Seq data. I extracted the regulated genes using DESeq2::results with a threshold of 1 ...
Hadeer Ibrahiem's user avatar
1 vote
0 answers
116 views

Working with Smart-seq3 data, have some questions regarding UMI and bar coding

Most of my experience has been working with genomic data, this time I am working on data from Smart-seq3. RNA excreted by cells in culture medium were processed with Smart-seq3 (From the looks of it, ...
Karthik Nair's user avatar
1 vote
1 answer
64 views

Filtering criteria for non-coding features with very low counts

I am trying to do DE analysis of non-coding features of A. thaliana. I find in the miRNA and lncRNA counts file that they are abundant in zero counts, and most of the non-zero counts are very low. Now,...
Arkajyoti Banerjee's user avatar
1 vote
0 answers
90 views

building custom gtf file with entrez ID for any organism

How do I generate a custom gtf file format for a organism of my interest similar to human gtf file, in the custom gtf file I want to add entrez id to the new custom gtf. My organism is ...
kcm's user avatar
  • 1,804
1 vote
1 answer
134 views

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 ...
Sam's user avatar
  • 149
1 vote
0 answers
172 views

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: ...
pythonbeginner's user avatar
1 vote
0 answers
22 views

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 ...
PesKchan's user avatar
  • 207
1 vote
0 answers
61 views

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 ...
Ahkam's user avatar
  • 11
1 vote
0 answers
48 views

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 ...
David's user avatar
  • 43
1 vote
0 answers
134 views

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 ...
RichardBJ's user avatar
  • 111
1 vote
0 answers
48 views

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 ...
h3ab74's user avatar
  • 846