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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1
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1answer
276 views

Spearman correlation between two genes

I have FPKM data for three genes like below: ...
4
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1answer
85 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
210 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
172 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
272 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
206 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
60 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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2answers
40 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
258 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
57 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 ...
2
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0answers
184 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
177 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 ...
0
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0answers
32 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
120 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: ...
0
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1answer
571 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
100 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 ...
3
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2answers
10k 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
49 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 ...
4
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1answer
138 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 ...
4
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1answer
41 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
154 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
56 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 ...
3
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2answers
894 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
82 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 ...
2
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1answer
134 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 ...
3
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0answers
196 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 ...
1
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1answer
654 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
29 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
87 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 ...
3
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1answer
102 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: ...
2
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2answers
39 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 ...
4
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3answers
503 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
107 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 ...
7
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1answer
75 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 ...
1
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1answer
48 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
108 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 ...
3
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2answers
692 views

Error creating indices using STAR

I am trying to index wheat genome using STAR through following command ...
1
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0answers
267 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 ...
0
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3answers
249 views

How to find correlation between two specific genes in same dataset?

I would like to plot the correlation between two specific genes in my data. I have a matrix with genes in rows and samples in columns, with read counts data. If I want to look at the correlation ...
3
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2answers
1k views

What is cellranger doing in comparison to other methods?

I've recently started working with the 10X-Genomics platform with Illumina (MiSeq and HiSeq) for single-cell RNA-Seq. I've been recommended the "cellranger" (version 2.1.0) which I understand handles ...
4
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3answers
240 views

Expression of a gene in different groups

I would like to check the expression of a gene in different groups like Disease vs Normal samples. I want to make a plot out of that to check whether it is significant or not. From this paper lncRNA ...
3
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2answers
87 views

What kind of analysis can be done with differential expression of transcription factors?

I have two different stem cell types and their respective gene expressions from RNAseq. I noticed that there is differential expression in some of the genes coding for histones as well as ...
0
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1answer
757 views

Transform and feed data into DESeq2 with DESeqDataSetFromMatrix

There is a normalized expression matrix. I split it into two and want to do DE on the two cells' subsets. I am having trouble ...
5
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2answers
433 views

Reads mapped to exonic, intronic and intergenic regions

After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason? ...
2
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1answer
76 views

How to get rid of the temp files while using “featureCounts” for extracting readcounts from bam files?

I used this command for extracting read counts for almost 60 samples using featureCounts. ...
0
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1answer
202 views

Average of logCPM

I am a bit in doubt how to calculate correctly the mean (average) in the logarithmic scale. I know e.g., that a division in the normal scale becomes a subtraction in the logarithmic scale, and so ...
3
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1answer
1k views

Subset on multiple genes in Seurat

I know that I can do subsetting on just one gene in Seurat: ...
0
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1answer
64 views

differentially expressed intron analysis?

I have used Deseq for estimating the differentially expressed introns for the RNA seq data using the UCSC intron bed file. can Deseq be used for differentially expressed intron analysis?
2
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1answer
2k views

clusterProfiler, groupGO: meaning of GeneRatio

I am running groupGO function from clusterProfiler: ...
1
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1answer
127 views

groupGO parameters explanation (ont and level)

I need to use groupGO in clusterProfiler to find functional profile of a list of genes, and I am having trouble finding out what ...

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