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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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24 views

How to quantile normalization on RNA seq counts

I have a read count data (RNAseq) and want to perform quantile normalization. Could you please help me how to do it. I tried some scripts in R but it didn't work. I want the result output in matrix ...
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1answer
43 views

data storage requirements for RNA-seq and WGS

Supposing upcoming RNA-seq for 3 conditions and 3 replications for each, how much is data storage requirements?
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2answers
116 views

Extracting some part of a list

I have two normalized gene expression values (log2 of cpm) ...
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1answer
37 views

Calculating Z-score from logCPM values using edgeR

I have the raw counts for RNA-Seq data. I converted counts data to logCPM using edgeR package. Lets say I have a dataframe A with 15000 genes as rows and 100 ...
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0answers
52 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: ...
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1answer
69 views
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1answer
25 views

log rank p value interpretation from gepia 2

How do i interpret the overall survival when the p value reported is not significant going by the standard convention . Not sure what kind of test it runs while comparing the expression may be one ...
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1answer
76 views

Changing this function to work

I have this matrix of expression data ...
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1answer
40 views

how to write italic script in Rstudio

I have got the problem on how to write the italic script in RStudio. I used the script in the screenshot and got the error "Error in grobs[[i]]: subscript out of bounds."
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50 views

Making a model of gene contribution in cancer diagnosis

I have raw read counts (Edgeseq technology) of 56 patients who have been ranked with Mandard score as responders and non-responders; I have done differential expression by DESeq2 and I obtained a ...
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1answer
29 views

Extracting genes differentially expressed by Wilcox test

I have 2 cell types (NOF and CAF) cultures individually and also have been co-cultured with tumour like this picture. https://ibb.co/x8Ty1Wz If this is the log cpm of these 4 samples ...
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1answer
59 views

Experimental Design for Differential expreression analysis

I have a Normal esophageal Fibroblasts (NOFs) cultured in DMEM media; The same NOF also have been cultured with a tumor sample from a patient named 005 on DMEM media; I have also Cancer Associated ...
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1answer
33 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 ...
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2answers
58 views

How can I extract the longest N isoforms per gene from a fasta file?

The question of how one can extract the longest isoform per gene from a multi-fasta file has been answered expertly in this thread: How can longest isoforms (per gene) be extracted from a FASTA file? ...
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35 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 ...
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1answer
63 views

Why is a PacBio read length larger than the aligned reference region?

I recently had some Iso-Seq sequencing done on my organism catfish on the new Sequel platform and got weird alignments for a size selected 4 Kilobase and up fraction after running the isoseq3 pipeline....
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2answers
41 views

How to link GDC ids to CCLE cell line names?

Hello bioinformaticians, I've recently downloaded few RNAseq data files from Genomic Data Commons Data (GDC) portal. These files belong to Broad institutes CCLE project. Now the problem is that GDC ...
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4answers
49 views

How to get the longest fasta sequence including all possible switching isoforms of a gene out of isoforms

I am working with RNA-seq data without a reference genome/transcriptome and am instead using a Trinity de novo transcriptome assembly. I analyzed both isoform and gene expression abundances using RSEM....
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1answer
25 views

Real time transcript profiles

Do any methods exist (or are in the process of development) for investigating transcript data without lysing the cells, i.e, destroying the sample?
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2answers
44 views

Why the t-test for a specific gene shows different value compared to differential analysis?

I have RNA-Seq data for LUNG cancer. 370 tumor and 50 Normal. For differential analysis initially I did some filtering and kept approx. 19k genes for further analysis. I used edgeR. With a FC > or &...
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1answer
41 views

How should I address batch effects in my experiment?

Let's say I have an RNA-Seq experiment, where I'm interested in the significantly differentiated genes between pre-treatment and post-treatment conditions. "rep" == biological replicate. ...
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1answer
43 views

Question about the dots on Quartile groups in boxplot

I have Microarray Normalized Expression data for a specific Gene. It looks like below in a dataframe B ...
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1answer
95 views

Integrative analysis of omics studies using machine learning

I would like to use public omics datasets (ChIP-seq, RNA-seq, and ATAC-seq) from different studies to do an integrative analysis as follow: Normalise samples, within each type of omics, from ...
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0answers
76 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 ...
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1answer
49 views

How to get the corrected matrix after SVA batch effect correction

I ran SVA to remove batch effects for my bulk RNAseq experiments, but now I need to somehow correct my data matrix in order to ...
2
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1answer
45 views

cDNA and alignment mapping

I am confused with RNA seq alignment. My understanding is that after Mature mRNA is isolated from the cell, it is then fragmented and using reverse transcriptase enzyme a cDNA copy is created which is ...
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2answers
64 views

How to deal with duplicate genes having different expression values?

I have RNA-Seq data which is FPKM. In the dataframe df first column is gene_name and the ...
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0answers
19 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 ...
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1answer
21 views

featureCounts segmentation fault on Arch Linux

I am encountering a segmentation fault when attempting to run featureCounts from subread-1.6.3 on even small test data. I installed featureCounts from SourceForge ...
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1answer
127 views

How to convert featureCounts to FPKM?

I have seen many posts regarding counts to RPKM and TPM. I haven't seen any post for counts to FPKM. I have RNA-Seq data which is paired-end reads. Extracted the counts using featureCounts for all ...
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1answer
36 views

How to calculate AUC in coverage graph

Is there a way to calculate the area under a curve in a coverage graph? Thank you in advance. EDIT: I'd like to calculate the AUC per strand per gene in a coverage graph. I have two wig files (...
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0answers
20 views

How to input data for DESeq2 from individual HTSeq count?

I am comparing the gene expression of 2 bacteria under 1 condition. I have now the count tables for 3 tech. replicates for each bacteria. ...
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1answer
61 views

Tool to remove a PCR contamination in NGS data

BACKGROUND I work on NGS data (illumina paired ends reads) coming from a full extract of RNA (metagenomic). We are interested in the viral fraction of this extract. I observed a contamination with a ...
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1answer
46 views

Why are my Chi-squared test results different from those in a published table?

I recently read the paper “A novel long non-coding RNA linc-ZNF469-3 promotes lung metastasis through miR-574-5p-ZEB1 axis in triple negative breast cancer”. In this I see Table1 showing correlation ...
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2answers
127 views

Seurat for clustering bulk RNA-seq?

Is it ever ok to use Seurat for clustering bulk samples? I am looking at FPKM data from ~750 bulk RNA-seq samples generated using Cufflinks. As suggested for FPKM ...
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3answers
102 views

Seurat Merged objects tSNE - How to paint on original IDs?

I am working with single-cell RNA-seq data, using the R package "Seurat" to cluster and visual data-points. I had two single cell datasets from which I generated two Seurat objects. I then combined ...
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1answer
58 views

Spearman correlation between two genes

I have FPKM data for three genes like below: ...
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1answer
64 views

Reference genome for allele specific expression

We are trying to sort out a pipeline for doing allele specific expression. Our plan is to call SNPs from RNA-seq data and combine with known SNP annotations. A well known problem in ASE is reference ...
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2answers
70 views

RNA velocity: competing explanations for variable ratio of spliced to unspliced transcript

In "RNA velocity of single cells", La Manno et al. look at ratios between spliced and unspliced mRNA as a way of estimating the velocity of a cell through transcriptome space. In checking for a ...
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1answer
46 views

Using Broad Institute RNAseq pipeline

There is a Github report for Broad's pipeline on RNA-Seq. It has a docker build file and WDL scripts. However, I don't see how any of the WDL script is being used in the docker (Dockerfile). It looks ...
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1answer
43 views

DESeq2 complicated design - effect of replicated samples

I have RNAseq data from a relatively complicated experimental design with variables = genotype, treatment, time, and batch. I have 2 biological replicates for each genotype/condition, however ...
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2answers
68 views

Find a cutoff value for genes that are expressed in single cell RNA-seq?

I want to find a cutoff value for each gene, above which we can consider a gene expressed. The problem is that not all effectively non-expressed genes will have 0 counts due to sequencing errors for ...
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0answers
23 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 ...
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2answers
31 views

K mean clustering issue

So I have expression data set with 4 healthy sample and 4 disease, to determine the number of K I used mclust which i ran on the list of differentially expressed genes so I got around 7 cluster from ...
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1answer
82 views

RNA-Seq: clustering/treatment of genes with low expression

I have some RNA-Seq data from leukaemia patients. I want to do unsupervised clustering on them with some other published leukaemia RNA-Seq data and see how they cluster. There are a few problems I ...
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0answers
38 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 ...
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0answers
37 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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1answer
44 views

Where to get '--fldMean' and '--fldSD' for single-end Salmon run

I need to run single-end bulk-RNAseq salmon - based alignment and I am not sure what I ...
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28 views

Quality checking benchmarks for clustering

I'm currently working with several RNAseq datasets from the ENCODE database. My approach to assessing the quality of the data, i.e, reproducibility is by automating the assessment of expression ...
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1answer
37 views

Generating list of known fusions for Arriba from COSMIC

The RNA-Seq fusion detection program Arriba features the ability to white-list known gene fusions, specifically the authors recommend using COSMIC as a source of these here. Specifically they state: ...