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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how to create "sample file" for the qAlign() function after trimming the reads in R

I'm an absolute beginner trying to solve this question "Align the trimmed and untrimmed reads using QuasR and plot alignment statistics, did the trimming improve alignments?" I did trim the ...
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IsoPlexis Data Analysis

Has anyone analyzed the raw data output from IsoPlexis secretome profiling? I want to make use of this data outside of their "end-to-end" software.
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What is the difference between Normalized Expression in EdgeR vs DESeq2?

I am trying to access the normalized expression in both edgeR and DESeq2, yet the results are different. Does anyone know why? How to get normalized expression using edgeR: ...
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baseMean threshold

I have an RNA-seq dataset and I am using DEseq2 to find differentially expressed genes between the two groups. I used pre-filtering to remove any genes that have ...
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Error in DESeq2 analysis with dimnames

I'm getting the following error in a DESeq2 analysis and I can't seem to figure out the issue: ...
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DimPlot - How to highlight cells with identity colors?

I made this wrapper for DimPlot: ...
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How will Seurat handle pre-normalized and pre-scaled data?

I want to use data from the following dataset: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84371 The data has been TPM normalized, which is not ideal for clustering but I have to work with ...
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RNAseq small sample size in control group

I am doing an RNAseq experiment with affected (n=6) and control groups (n=6) in cow. However it turned out that 4 of my control samples have very low quality so I have to discard them. My problem is ...
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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 ...
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Target genes for piRNA

Where I can find a database or tool to give me the target genes of PIWI (piRNAs) in human? I found one but works for worm like <...
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RNAseq SNP discovery: deciding upon filters and dealing with allele expression bias

I am working with non-model plant RNA samples which we have been deep sequenced and analysed using STAR aligner under default parameters. Aim We would like to conduct SNP discovery of these samples. ...
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Workflow for identification of new splicing variants from RNA-seq data?

I'd like to perform a search for possibly unidentified splicing variants of a specific protein in A. Thaliana. I do not have my own RNA-seq data. It has been suggested to me to use this workflow: ...
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Trinity de novo assembly not completing

I'm trying to assemble some transcriptomes using Trinity, but am having issues getting Trinity to finish. I've been trying to get this to work for weeks, but am hitting a wall and would very much ...
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Low mapping reads for RNA sequence

I am new to RNA sequencing analysis. I have RNA samples for bacteria for different treatment. I did contaminant filtration, remove adapters and checked fastQC report: attached below for one sample (...
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A two color bar plot in R

I have raw read counts of miRNAs in two conditions like below ...
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subsetting more than one ccle sample

I'm working with the CCLE dataset and I'm trying to subset the data to just 3 cell lines of interest but not sure how to it. Currently, I have been subsetting one by one but is there an easier way? <...
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Why is bulk RNA sequencing reflecting AVERAGE expression but not TOTAL expression of all cells?

When I am reading papers that compares bulk RNA sequencing and single-cell RNA sequencing, we often see papers describe bulk RNA seq measures the average cell expression. For example, in this paper ...
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Pulling normalized gene expression data across cells from Seurat object

I want to pull gene expression out of a seurat object. I created one called combinedObject following their tutorial, I normalized scaled it etc and created some ...
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2 answers
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Plotting a gene in Seurat

I saw in the extensive Seurat documentation for Dimplot (dimensional reduction plot), here, you can plot a gene by specifying it with group.by = "gene" but this does not work in practice. <...
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Comparing differential expression across samples - is batch effect correction needed?

I have a bulk RNA-seq dataset made up of control and treatment conditions for a range of cell lines. This dataset was generated in two batches, such that the cell lines are split between batches but ...
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Normalization methods for single cell RNA sequencing that take read count into account

I have two RNA-seq datasets. One was sequenced at an average read count of 1.5 million per cell the other at 43K average reads per cell. For the first I also have the meta data from reads alligned ...
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2 votes
1 answer
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Best way to match genes in two RNA-seq count matrices from two geo accessions

I have downloaded two datasets, and I am trying to remove the genes they do not share in common. Is there a biocmanager package with a function that can compare their gene ids, match them and reorder ...
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Is it possible to do cell composition for scATAC-seq data without scRNA-seq data?

Background My understanding was that if I did scATAC-seq and I have some clusters of cell groups, the only way I can label it is by correlating those groups with scRNA-seq data. My lab ordered some ...
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What could explain the difference in RNA-seq and Microarray expression levels?

What are some possible reasons why some trends observed in Microarray expression levels is not observed in RNA-seq. Example the difference between 2 cell types for a gene of interest show major ...
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TGCA LAML rsem data

How do I test the gene expression differences between multiple groups? I have normalized TCGA RSEM data, this is the dataset ...
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What does the last column of segemehl's .trns.txt file mean?

Segemehl creates .trns.txt, a "custom text file contains all single split alignments predicted to be in trans, i.e. split alignments that are located on ...
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How to solve correlation problems between different samples in scRNA-seq?

I am trying to align and merge different samples from NCBI. I end up having correlation problem with these sample. The picture below shows an heatmap of the R² by doing a linear regression between 2 ...
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Some definitions about RNA-seq

I want to select a 10x single cell kit What does 2x 50 75 100 150 250 mean in paired end sequencing?
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How to input data and metadata from NCBI for RNA-Seq analysis in R

I am quite new to R programming so I hope my question is a clear one and not too confusing. So, I have downloaded some .txt and .csv files containing the raw count data (I am supposed to find out ...
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How does DESeq2 "collapseReplicates()" function work on read counts data?

Comparing read counts from an RNA-seq experiment for two select genes before and after using DESeq2's collapseReplicates() and ...
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How to remedy a DESeq2 collapsing technical replicates error?

Goal: To ensure "the sum of the counts for [my samples] is the same as the counts in the [samples] columns in ddsColl" after collapsing replicates using ...
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How can I get a column with real gene names in my ballgown analysis?

I am doing RNAseq analysis and I am using ballgown procedure for it. There is an option in R to calculate differentially expressed genes and use FPKM in calculating differential gene expression, which ...
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Should I remove days_to_death = 0 before TCGA survival analysis

Some patients in TCGA data have days_to_death = 0 and days_to_follow_up = NA vice versa. Should I remove these samples before survival analysis? What does days_to_death = 0 mean? Does it mean the ...
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Nextflow HISAT2 command exit status 255 with prebuild index

I have a problem with my nextflow pipeline. My workflow looks like this: Uncompress the hisat2 genotpye index grch38_genome.tar.gz from https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz ...
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How to find an unidentified splicing variant of a protein?

I have some data indicating there might be a splicing variant of the Arabidopsis Thaliana protein I'm studying that has not been identified. Is there a database of i.e. RNA sequences (transcriptome?) ...
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Why use shuffled input-sequences as background of MOTIF discovery? (from CLIP-seq data using MEME-suite)

By default, STREME (MEME-suite) uses second-order shuffles of the input sequences as background. STREME highlights its usefulness for CLIP-data, which would be my origin of sequence-inputs. In this ...
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Why does BWA MEM orientation contradict my library prep method

I have some RNA-seq data from a stranded paired end library prep, with dUTP and UDG preparation, so the orientation should be RF (confirmed with sequencing provider). I assembled the reads with ...
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how can I get expression of an inserted foreign genes?

hi we have transgenic mice with human gene inserted. if we profom rnaseq for the mice, how can we get the expression value of the human gene inserted to the mice? and use this expression to compare ...
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Gene expression changes in one condition

I want to look at an RNA-seq data-set on 1 condition to look for expressed genes. As there is only one condition, I can’t perform differential analysis. The only ...
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1 vote
2 answers
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SyntaxError after end of Snakefile code?

I am trying to run Fastqc on multiple files using Snakemake but am receiving an ambiguous SyntaxError past the last line of code in the Snakefile. Snakefile: ...
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Snakemake Fastqc: "Multiple run or shell keywords in rule run_fastqc."

I am trying to check the quality of RNA-Seq data from Illumina using fastqc in snakemake in a conda environment. I get the error "Multiple run or shell keywords in rule run_fastqc". ...
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What is the meaning of multiple "cases" in GDC data files

I thought I had a good handle on the data model of GDC, but a recent query on the TARGET dataset revealed something I had not seen before. TL;DR- what does it mean when multiple "cases" are ...
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1 vote
1 answer
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Best R program to use for RNA seq analysis

We have an RNA seq taken from a the same tissue in two different developmental stages. We have one gene, with the number of reads from stage A and stage B, and we want to see which genes are ...
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How to simulate replicates for DGE analysis?

I am prototyping with data visualization of DGE results, and I would like to work on the analysis pipeline before the real data is available. Currently, I only have 3 samples for wild type and 1 ...
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Help with performing PCA analysis on data from 3 different datasets of normalized counts data for RNA-seq experiment

So, I have limited knowledge of R but I need to do a PCA analysis of 3 different datasets of gene expression as a result of combined growth or mono-culture growth. The 3 different datasets I performed ...
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1 vote
1 answer
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Error to running "CIRIquant" function in CIRIquant package at python Language

"CIRIquant" is a Python package for detecting Circular RNA. I installed "CIRIquant" through the below command in terminal: pip install CIRIquant My Linux distribution is Ubuntu ...
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1 vote
1 answer
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Different contrasts in DESeq2

I have a treatment and control in two time points like this ...
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1 answer
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Convert VCF file to mpileup.txt

I am working on an iterative analysis that uses orthologous pipelines that require mpileup.txt files as input for a visualization step. This requires me to convert VCF files to mpileup.txt. This ...
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3 votes
1 answer
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Alternatives to CAP-miRSeq for microRNA processing

I am involved in a Drosophila transcriptomics project in which we have RNAseq data for small RNAs (for microRNAs) in addition to total RNA (for mRNAs). The total RNA reads have been analysed ...
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Why is bcl2fastq2 taking so long to calculate stats?

Our lab has been using bcl2fastq v2.20.0.422 to demultiplex RNA-seq data sequenced on an Illumina Novaseq machine on a beefy EC2 instance and we've run into the strange problem: namely that while ...
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