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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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1answer
73 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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46 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 ...
0
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1answer
18 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
43 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
54 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
14 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 ...
1
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1answer
20 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 ...
0
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1answer
49 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 ...
0
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1answer
30 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
17 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. ...
2
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1answer
46 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 ...
3
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1answer
41 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 ...
3
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2answers
79 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 ...
1
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3answers
49 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 ...
1
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1answer
46 views

Spearman correlation between two genes

I have FPKM data for three genes like below: ...
4
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1answer
55 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 ...
2
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2answers
34 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 ...
0
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1answer
37 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 ...
1
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1answer
38 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 ...
1
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2answers
59 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
20 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
30 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 ...
4
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1answer
66 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 ...
3
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0answers
35 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
30 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
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1answer
28 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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0answers
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 ...
2
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1answer
33 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: ...
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1answer
87 views

Batch Effects in RNA Seq Sample

I am using the R (using EdgeR) for the RNA Seq analysis, I had few batch effect samples like Control vs treatment. Could anyone tell me the best way to remove the batch effects. I have looked into ...
3
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1answer
41 views

Change sample names while using cummeRbund

I want to analyze RNA seq data using R package cummeRbund. Using this package I have a problem changing sample names. When I run this command in cummeRbund ...
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2answers
220 views

How to scale the size of heat map and row names font size?

I have an expression data matrix (120X15; 15 samples and 120 genes), my heatmap looks blurred and raw names (gene names) looks very small and can not read. How can I improve my scripts? Here is the ...
2
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0answers
27 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 ...
3
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1answer
69 views

Plot to show the expression of genes between tumor and normal

I have RNA-seq raw counts data for 50 samples. 20 Normal and 30 tumor. After differential analysis I got 30 DEGs. I want to make a violin plot showing the expression of each gene. I transformed counts ...
2
votes
1answer
30 views

size of the pathways for analysis and filtration

I have recently started working on a substance's effect on a cell line in different dosages. for this, there is a tool called bmdexpress2 that I am using. Its input is the normalized counts from ...
2
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3answers
92 views

Selection of differential expressed genes

I'm working with RNA-seq data. I have 40 tumor samples and 5 Normal samples. Differential analysis with Deseq2 based on Fold change > 1.2 and alpha < 0.05 gave ...
2
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1answer
30 views

RNA seq fasta file annotation from alignment to reference matches

I've got a fasta file with some RNA seq data and another csv file with the output from plast where I've aligned it to a reference using plastn. I'm struggling with figuring out a command to append my ...
2
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2answers
119 views

gene-level versus transcript-level analysis

Traditionally, RNA-seq data was quantified on gene level. Newer methods quantify on transcript/isoform level. For example, Kallisto only outputs transcript-level abundances. From the DESeq2 vignette: ...
0
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1answer
54 views

Why is FPKM still used for gene expression studies?

Perhaps my understanding is wildly misguided, but I'm seriously confused about why people are still interested using FPKM values for cross sample gene expression analysis. My understanding is, that ...
0
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1answer
60 views

Negative scale-free topology

I am trying to build gene co-expression networks with WGCNA by combining in-house and publicly available RNA-seq datasets. I am interested in identifying gene networks associated with different ...
2
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0answers
52 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 ...
0
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1answer
88 views

How to calculate logCPM across all samples?

Using edgeR for differential analysis between Tumor and Normal gave me differential expressed genes with logFC, logCPM, PValue and FDR. From the details of glmTreat function I see that logCPM is ...
0
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1answer
26 views

What are some good sources of 3rd order gene expression data

I need good sources of gene expression data in the form of 3rd order tensors. Typically the commonly available datasets are in the form of a matrix, for instance, $sample \times gene$ or $gene \times ...
4
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1answer
40 views

Is removing samples based on clustering for downstream analysis a right choice?

I'm using TCGA Lung cancer data. I'm interested in doing differential analysis between Lung vs Normal. Before DEA, to check the distance between each pair of samples I plotted an MDS plot: In this, I ...
2
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1answer
46 views

Detecting differentially expressed genes with foldchange >= 2 and FDR < 0.05

I'm using edgeR for differential analysis. Using glmTreat function I'm detecting differentially expressed genes between Tumor and Normal. I have set the arguments like below: ...
3
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2answers
33 views

Can I ask DESeq2 which variable alone explains which gene's behavior the best?

Imagine I have a dataset of highly periodic gene expression, taken at densely spaced timepoints. I know that the expression is periodic, but different genes have different offsets. It is trivial to ...
3
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2answers
152 views

Issues performing variant calling with GATK

I am trying to perform variant calling on a BAM file generated through STAR version STAR_2.6.0b for wheat genome using GATK haplotypecaller as follows: ...
1
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1answer
44 views

How to search a specific sequence in BAM files for 10X experiment

I have to search a specific sequence in a set of cells (> 1k cells) from a single-cell experiment done with 10X genomics. As input file I have a single bam file, and 24 fastqs, therefore each file ...
6
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1answer
49 views

Network comparison of single cells (from sequencing data)

What methods can we use to compare networks (PPI, gene regulatory etc) created from single-cell gene expression (or proteomics) data? There are methods that construct networks by comparing two ...
0
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1answer
37 views

Retrieving gene matrix from internet in GSEA desktop

This error appeared to me, and I have the program connected to the internet. I already installed java 8, but it still doesn't get me the gene set databases.
5
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2answers
55 views

Smallest group size for differential expression in limma (bulk RNA-Seq)

I am reading Smyth et al. (ref. 1). I want to run differential expression analysis on a bulk RNA-Seq dataset in which each group is composed by 2 samples. In the paper previously cited it is written ...