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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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45 views

RNAseq data analysis

I am currently doing a Differential Gene Expression of RNAseq data in Lung Adenocarcinoma using TCGAbiolinks. In the data Preprocessing step TCGAanalyze_Normalisation, I am confused as to which method ...
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39 views

How I deal with this expression set?

I have a gene expression raw counts like below for 16 patients ...
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24 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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23 views

Tree cut issue in WGCNA

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37 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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2answers
100 views

too long header for fasta file

I have a fasta file, like this: ...
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0answers
45 views

Selecting genes with more contribution from PCA

I have RNA-seq data in response to treatment vs non response; By machine learning I selected three principle components likely can predict the response based on the gene expression. Now I have ...
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1answer
37 views

Convert FPKM to RPKM

I have my 24 samples analyzed by Hisat2>Stringtie, from paired end sequenced data. I have got FPKM data and am planing to convert them to RPKM. to be able to compare my samples with another already ...
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1answer
17 views

How to map selected genes to Metabolic pathway Maps

I have a selected Arabidopsis Genome Initiative (AGI) list for RNA seq and proteomics data, how can I map them to metabolic pathway maps to vilualize(e.g. TCA cycle / FA / Photosynthesis) in KEGG or ...
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1answer
60 views

Changing the axis limits of ggplot objects

By EnhancedVolcano R package I have this plot with this code ...
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1answer
79 views

How I deal with this kind of gene expression comparision

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example tumor 1 in batch 1 and tumor 1 in batch 2 , ...
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2answers
66 views

TPM or rlog(CPM) for comparing expression?

I want to see the expression of a gene in a group of patient amongst the entire cohort using my RNA-Seq data. While I can do a differential expression analysis with limma or DESeq2, I want to see how ...
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2answers
27 views

In-sample and across samples normalized expression

I want to get the expression data that is in-sample normalized like FPKM and also across samples normalized as obtained using DESeq2 or else. What I am currently doing is that I first normalize the ...
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0answers
28 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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2answers
77 views

Interpreting this PCA plot for RNA-seq

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example ...
2
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2answers
48 views

Find most similar genes to a set of genes of interest in time series RNA-seq data

I have gene expression data (RNA-seq) for 30 different time points (from 0 to 60 min each 2 min). I have a set of 8 genes that behave similarly (although not identically) and I want to find the top X ...
2
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2answers
57 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
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1answer
30 views

RNA-seq: How to get new expression count after normalization

I've RNA seq, Human, Paired-end data, Sample size is <40. These are aligned using STAR, RSEM processed. With RSEM I've TPM and expected counts, that is two files columns as individual IDs and row ...
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0answers
44 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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27 views

how to cluster data separately based on HTO tags

If I have 2 samples hashed, ran together and generated one ADT file for them, then how do I plot 2 different ADT clusters separated based on the hashtags. I did use HTOdemux, but it shows me ...
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1answer
39 views

How do I create a filtered gene list using expression medians

Forgive the simple noob question I have TPM data of ~50k genes (rows) across ~1k cell lines (columns). In R, I would like to output an "intermediate expression" gene list for each cell line, like: <...
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0answers
22 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://...
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13 views

What Galaxy tools can annotate a de novo assembly RNA seq plant transcripts?

What Galaxy tools can annotate a de novo assembly RNA seq plant transcripts? How to do it using Python API?
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1answer
53 views

After running a DRIMSeq pipeline, how do I know which genes are upregulated in the different conditions?

After running a DRIMSeq pipeline and obtaining the genes that are differently used between the null and full models, how do I select the genes that are differently used in the different conditions? ...
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1answer
50 views

Comparing RNA-Seq and Ribo-Seq

I'm attempting to compare total RNA-seq with Ribo-Seq, to determine if changes in Ribo-Seq are due to changes in transcriptional expression (akin to the analysis performed by Anota2Seq). I am, however,...
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0answers
42 views

Identification of differential genes across 8 groups [closed]

I need to identify differential genes across 8 groups. I know this can be done using DESeq2 or EdgeR as these are better suited and equipped. However, I was thinking if something like this piece of ...
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1answer
69 views

Correlate DEGs from DESeq2, EdgeR and Limma results

I have a lists of DEGs identified by DESeq2, EdgeR and Limma. I would like to correlate the the gene rankings in the lists to decide on a package to use in downstream analysis. I am havig a few ...
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0answers
39 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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1answer
74 views

How to transform and sort the matrix to make a heatmap showing signatures?

I have a matrix data with cells as rows and samples as columns. Here I am giving the data with dput ...
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1answer
54 views

design formula question

I am not sure I am building the proper design formula for the question I want to answer I have the following samples with three factors; clone, the structure and the condition. ...
2
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1answer
40 views

Single Cell RNA seq Analysis for low input Cells?

I am having a single cell RNA seq data from ~500 Cells. I do understand this number is very low for present day analysis tools like Seurat. So, I want to know what if one have a such a small number of ...
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1answer
53 views

How do I select a proper design matrix to use in DRIMSeq?

I am trying to use DRIMseq for DTU with 2 treatments on two different strains of an animal model. How do I select a proper design matrix to use in DRIMSeq ? How I know that one is the correct one to ...
2
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1answer
141 views

How to find novel transcripts using GFFcompare?

I am trying to find novel transcripts from an RNA-seq database. Based on the advice I got, it seemed that using Stringtie for transcript assembly is a good way to go, and it supports novel transcript ...
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1answer
97 views

What are the right parameters to trim a small RNA transcriptome with trimmomatic?

I'm having some problems with finding the right parameters to trim my small RNA Illumina reads (51 nt long) with Trimmomatic. Before trimming, one of the samples (21M reads) looks like this: So for ...
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1answer
57 views

Cluster is split in 2-3 locations on tsne plot - Suerat

I am running a single cell dataset (count data - exon) through Seurat. After running tsne I see a cluster (13) split in 3 different locations on the plot. Here are the commands I am running: ...
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2answers
101 views

Strange p-value histogram for differential gene expression analysis

I'm trying to perform pretty standard differential expression analysis using RNA-seq data. I've used Kallisto to perform RNA quantification and am using Sleuth to perform the differential expression ...
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0answers
79 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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0answers
34 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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0answers
21 views

Normalization for microbiome 16s sequence analysis

The way I understand things, normalization (such as in DeSeq2, EdgeR, etc.) serves two purposes: 1) Model the "real" abundance in the original samples from the read counts, 2) Make the abundance ...
0
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1answer
39 views

Why does DESeq2 convert numeric columns to factor during differential expression analysis?

I'm attempting to perform differential expression analysis using DESeq2 on a dataset of ~90 samples and ~20000 transcripts. I have a number of variables I'd like to test against, some of which are ...
1
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2answers
193 views

How drastic would the result be if I use fasta or reference assembly from ucsc and gtf from gencode?

There are difference annotation file for UCSC and gencode. But if I use the reference assembly from UCSC and the GTF from Genocode or vice versa would my downstream results would be wrong?
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0answers
51 views

Normalization of data with rpkm

I'm very i difficult with normalization of my data. I was searching for transposable elements in my genome, and after this step, I made counts of reads in some transcripts. I produced something like ...
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1answer
45 views

how can i download rna-seq data sample for starting rna-seq analysis by R? [closed]

I am looking for an induction into RNA-seq analysis using R. I have yet to decide a data set, or a subject area. Any help in this regard would be welcome.
1
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1answer
27 views

bedtools feature out of bounds

I'm trying to count the GC in regions leading up to polyA clusters. I found the bedtools command nuc, one of the outputs of which is the GC content. I took the mouse polyA cluster database from here,...
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2answers
91 views

Can we use GTEx data as control data for TCGA data?

I am using Recount2 TCGA data and was wondering is it right to use GTEx data as control data for this. I would really appreciate your views on this?
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1answer
114 views

bowtie2 options when mapping stranded single end reads

I'm trying to map RNA-Seq reads generated using the NEB Ultra Directional kit is first strand reversed protocal. I'm using bowtie-2.3.5_1 to align against the mus ...
1
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1answer
162 views

Align legends to each heatmap in a ComplexHeatmap with 2 heatmaps

I have created a ComplexHeatmap containing 2 Heatmaps, each with their own legend. The legend on the first Heatmap is continuous, whereas that of the second is ...
1
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2answers
142 views

GSEA - Gene Set Enrichment Analysis - in R studio

I have been trying to conduct a classic GSEA using the broad institute software and can not figure out how to compile it in R for the life of me. I am aware that there exists other packages in R that ...
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3answers
82 views

Calculating p-value for introns which are retained (or not) between 2 conditions

I am trying to calculate p-values and ultimately FDR values for RI introns for 2 conditions of experiment I have(control and knockdown) For every condition I have 3 biological replicates each: 3 ...
7
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1answer
315 views

Why are my kallisto and salmon results differing so much just for lncRNA transcripts?

I am running some analysis on an RNA-seq dataset. I have a list of transcripts that are potential lncRNA for which I ran both Kallisto and Salmon aligners. The input data for index building and ...