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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50 views

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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1answer
12 views

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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38 views

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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2answers
51 views

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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1answer
42 views

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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0answers
13 views

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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1answer
59 views

Best R program to use for RNA seq analysis

After performing RNA-seq, I am left with a CSV file containing a list of genes and the amount of reads in two different cell types. What is the best package to analyze the data for significant ...
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0answers
16 views

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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1answer
64 views

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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1answer
30 views

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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1answer
89 views

Different contrasts in DESeq2

I have a treatment and control in two time points like this ...
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1answer
37 views

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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Alternatives to CAP-miRSeq for microRNA processing

I am involved in a Drosophila transcriptomics project in which we have been obtaining RNAseq data for microRNAs as well as mRNAs. In initial work we used CAP-miRSeq for the latter as the Tuxedo ...
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41 views

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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35 views

How to perform a meta-analysis using data consisting of paired-end and single-end reads generated from Illumina and Ion Torrent?

So basically I have RNA-seq reads that were generated from Illumina and Ion Torrent platforms for yeast species. I have seen an article where they compared liver cells of a rat that were sequenced ...
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2answers
79 views

Is it possible to do DEG analysis without replicates?

I have three bulk RNA-seq samples (test1, test2, test3) without replicates. And I noticed that DEG analysis tools such as DESeq2/edgeR/etc cannot be applied for data with no replicates. So, I just ...
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1answer
38 views

Assembling all transcripts for an individual gene? (using single sequence to seed the assembly)

Let's say I have a candidate gene and I believe that in an individual sample, the genome sequence differs from the reference which then interferes with alignment. Is there a way for me to do a "...
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16 views

java error when running CIRI-full.jar Merge module

I have an issue running a module trying to detect circRNA from RNAseq data. The error is a java error and I have not knowledge of java so I don't understand the issues and googling hasn't helped. the ...
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21 views

A very large number of clones in BCR reportorie

I am using mixcr to convert fq.gz raw data file (single cell BCR sequencing) to txt files with the names such as JX01_d3-B.clonotypes.IGH.txt , Then I use the immunarch to load the file to explore the ...
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2answers
54 views

Counting the number of gene isoforms in a GFF3, is this method correct?

Recently I've been tasked to count the numbers of gene isoforms for each locus in a .gff3 file. I'm still doing my first steps in biology and bioinformatics, so I ...
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1answer
37 views

Error when using awk command to trim sequence files

I am attempting to edit my fastq sequence files with an awk command from a published article. The sequence files are all ...
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0answers
64 views

Splitting .bam files into separate samples for tophat2

I have RNA seq data of 12 samples (paired end, so 24 files) and I ran tophat2 for alignment on all of them which gave me one bam file for all the samples. Now I need separate bam files for all the ...
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1answer
51 views

Limma decideTests function: what kind of multiple hypothesis testing correction does parameter "method" involve?

What kind of multiple hypothesis testing correction does method="global" do in Limma's decideTests function? According ...
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0answers
24 views

Gene co-expression network analysis

I am working on a genome-wide study and struggling with gene co-expression network analysis. I am working on Tomato(Solanum lycopersicum). Can someone suggest me some tools or methods for this ...
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1answer
30 views

Keeping rows with a cut-off in all columns

I have a data frame of raw read counts, genes in rows and samples in columns ...
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1answer
67 views

shiny dashboard to visualise RNA seq data

I am trying to build a shiny dashboard for the visualization of different RNAseq data sets. However, I am encountering problem with reactive input, the code is always using the second dataset that I ...
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1answer
80 views

Beginner with DESeq2 having issue with analysis

Hi there I am brand new to using RStudio and trying to run DESeq2 analysis on my featureCounts output counts table. I ran the following code: ...
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1answer
31 views

Parallelizing microRNA targets

I am trying to look for miRNA targets using a file called Conservedfamily.txt from the Zebrafish target scan fish website. I have written a python program to ...
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1answer
51 views

DESeq2. Which is better: using the altHypothesis argument in the results function or manually filtering for your desired P-value and Log2FoldChange?

In DESeq2 you can identify your differentially expressed (DE) genes using the results function. I noticed people in my lab using the results() function with the minimum number of arguments supplied (...
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3answers
117 views

The confusion of using TPM (transcripts per million)

It is shown that TPM values are not suitable for DEG analysis but good for within-sample comparison since TPM normalized the gene length. My question is first: if TPM is not suitable for across ...
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0answers
10 views

Taiji doesn't run Pagerank

I'm trying to use Taiji to run a pagerank on our combined bulk RNAseq and ATACseq dataset. Here's how we run it: taiji run --config config.yml -n 10 Here's ...
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1answer
65 views

how to get normcounts for singlecellexperiment object?

I need to perform differential expression analysis using the scDD package from R, but I am not able to since I miss the normcounts assay in my SCE object (of course in the example they show, the assay ...
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1answer
50 views

Understanding "Measurement of mRNA abundance using RNA-seq data" by Wagner et al 2012

I'm trying to understand why RPKM is not an appropriate way to normalize for RNA seq (I understand the general idea, but I'd like to gain a deeper understanding). So I'm reading the original paper by ...
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47 views

Correcting for batch effect in bulk RNA seq datasets

I have samples from many different bulk RNA seq studies that exhibit an evident batch effect (samples from different labs cluster together regardless of treatment). I've found a lot of papers (here, ...
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1answer
22 views

Combining read counts from three separate GEO studies

I want to do differential expression analysis with DESEQ2. I have three read counts files downloaded from GEO (small RNAseq based) where the number of miRNAs and id is nearly the same. These studies ...
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1answer
72 views

Batch effect correction using a subset of samples - using DE genes

I have some RNA-seq data with two very obvious batches as you can see in the PCA: The samples of interest (A - H) are from tumor tissues. In addition, there are data for two cell lines I and J. The ...
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0answers
16 views

Installation of software Capmirseq

I am trying to install a software called Capmirseq. After installing all relevant modules I followed the installation instruction code as follows ./install.sh -p /path/to/software. Which is expected ...
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1answer
45 views

RNA_Seq data aligned used uniquely or multi mapped reads impact on result interpretation

I have some transcriptomic (Whole) sequencing data that I should analyse. I would like to do raw data alignment to a reference genome taking into account the multi mapped reads and uniquely mapped ...
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0answers
12 views

Ccle miRna data units

Does anybody know what units the mirna expression data on ccle is in? I saw the pipeline in the original paper but it doesn't mention units. Original paper: https://www.nature.com/articles/s41586-019-...
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0answers
41 views

How to create a DESeqDataSet and define experiment design before varianceStabilizingTransformation?

I have an RNAseq count matrix consisting of 2 groups (high, low) with 6 timepoints per group (T1,T2,...,T6) and 3 replicates per timepoint (rep1, rep2, rep3). So a 2-factor design with 36 samples in ...
2
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1answer
53 views

How does FDRtool work?

I have a question about using FDRtool. In the below code (on RNA seq data whose p values were acquired using Deseq2), the FDRtool was first used and thereafter p.adjust using the benjamini hochberg ...
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2answers
178 views

Calculating most abundant transcript from RNA-Seq data

vcf2maf uses VEP to annotate variants, and I believe selects the default Ensembl transcript to use for annotation. Sometimes the ...
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1answer
55 views

Get Salmon mapping/alignment summary

With HISAT2, after the alignment of fastq files you get an alignment summary like this: ...
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1answer
50 views

Base abbreviations other than ACTGU in sequence file

Short question: where can I find what F and J and < and ...
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1answer
75 views

What is the meaning of split read?

I want to use rna seq data to later perform functional tests on fusion genes. so before that I need to filter the "best results" (of rnaseq) for deciding which candidates I actually want to ...
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1answer
199 views

What are common ways to calculate gene length for TPM calculations? [duplicate]

I am aware of this similar question. But the accepted answer there answers how to calculate TPM given a mapping from gene name to gene length. My question is, given an annotation file of genes (for ...
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2answers
49 views

RNAseq DE comparison with samples of different read length [duplicate]

I have RNA samples but with different read lengths (Eg, HiSeq 2x125 and NovaSeq 2x150bp data). I would like to do DE analysis on these samples. What do you recommend? Do you recommend to trim the ...
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5answers
161 views

Normalize RNA seq data from multiple runs for expression analysis

I have RNA samples sequenced with TruSeq Stranded Total RNA kit protocol in Illumina HiSeq (2x125bp) and NovaSeq platforms (2x150bp) - almost 100 samples altogether. I have to use the samples data for ...
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1answer
49 views

correlation coefficient versus DEGs analysis: what's the best approach for low expressed genes?

I have 6 folders. Each one contains 7 datasets of a specific type of cancer (RNA-Seq) and 7 datasets of normal tissue (healthy control for that type of cancer). A total of 84 datasets. I want to ...
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0answers
26 views

Data format for pathway based clustering of samples

I came across this paper as one of the examples from this paper, this one Figure 2. Host Protein Alterations in Infected iAT2s ...

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