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
19 views

Cuffmerge: [Samtools-help] EOF marker is absent. The input is probably truncated

I need to merge my all transcripts.gtf from cufflinks output follow this command line : cuffmerge -o merged_gtf_output -p 15 -s ref.fasta -g anot.gtf assembly.txt but I have this warning : [...
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22 views

Coding object structure properties into a sequence

Hopefully, I found the right place to ask. Please, note that I'm not a specialist in the current field. Is there an algorithm to code information about object structural properties into a sequence (...
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13 views

Filtering genes from cuffdiff results

I have run cuffdiff (with statistics turned ON) to compare two groups of samples: Control group and Late AD group. This is the command I ran to be precise: ...
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2answers
29 views

Is there a computational tool or possibility to identify mRNA isoforms from the count matrix of a bulk RNA sequencing dataset?

I have the counts matrix of an RNA sequencing dataset of fibroblasts and I wish to identify isoforms of a particular gene of interest in it. Can anyone please hint me on a bioinformatics method to ...
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1answer
33 views

How do I differentiate outliers from in-group variations in a DESeq2 PCA plot of 9 samples distributed into 3 conditions?

I have a PCA plot from DESeq2's plotPCA(vsd, intgroup=c("conditions")) function. I have 9 samples distributed in to 3 groups of 3 biological replicates each. My ...
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29 views

RNAseq without depletion?

I have been asked by my supervisor to conduct an RNAseq without experimentally depleting the samples. However I am quite unsure regarding how much sequencing depth per sample I would require in order ...
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1answer
33 views

How to calculate fold change in gene expression from RSEM (RNAseq)

I have gene expression data from RNAseq, specifically: log2(x+1) transformed RSEM normalized counts. How can I convert these data from multiple samples to determine fold change in gene expression?
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1answer
27 views

Download sequences of isoforms

I want to collect all isoforms of all genes (Fasta/Fastq file of nucleotide and protein sequences) Arabidopsis Col-0. I am wondering if there is a straightforward way to download the file from any ...
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1answer
30 views

How to run trimmomatic in HPC

Sorry if this has been asked before, I've done a quick search and I don't think there's an easy explanation for me to understand. I'm really new to bioinformatics/RNASEQ analysis, having only taken ...
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1answer
42 views

How are these definitions related to differential expression?

I have two groups of patients; for each patient I have an output file (RNA-seq) contains this information ...
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2answers
47 views

Importance of Proper Pairs vs Aligned Reads for RNASeq data

I have stranded, paired end RNASeq reads that I have aligned using STAR. I plan do conduct a differential expression analysis with DESeq2. After running quality control checks, a good portion of my ...
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2answers
117 views

STAR quantMode vs RSEM vs Kallisto

I recently discovered this Snakemake pipeline for RNASeq that uses STAR's quantMode to quantify gene expression for DESeq2 differential expression analysis. In the past I've always seen the workflow ...
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2answers
174 views

How to initiate RNA-Seq analysis of TCGA files?

I want to compare RNA-Seq datasets obtained from the TCGA to investigate how my gene of interest is implicated in different types of cancer. I'm trying to download the data from the GDC Data Portal (...
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33 views

Simulating RNA Editing

I'm pretty new to bioinformatics and I have been looking for a way to simulate rna sequencing with editing, specifically A to G, but didn't find anything i could use. I tried using flux simulator but ...
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1answer
54 views

How to visualize called narrowPeak files in UCSC Genome browser or IGV?

I have called peaks using MACS2. Then I got a narrowPeak file like this. ...
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1answer
39 views

hisat2 --rna-strandness option and downstream htseq-count analysis

I've got some doubts on the hisat2 --rna-strandness option and its output for downstream analysis. Please see below. I understand that the --rna-strandness option produces an XS tag to indicate where ...
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5answers
7k views

Why does the FASTA sequence for coronavirus look like DNA, not RNA?

I'm looking at a genome sequence for 2019-nCoV on NCBI. The FASTA sequence looks like this: ...
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1answer
17 views

Error in createMAE function: non-unique values when setting 'row.names' in TCGA LIHC Data

I am working with the createMAE() function of the ELMER Bioconductor package. While executing the createMAE() function using ...
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0answers
13 views

Programmatic Access to `fastq-load.py` Arguments in SRR Metadata

Is there a way to programmatically access the fastq-load.py arguments from the SRR metadata? Specifically I would like to capture the exact command line arguments ...
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1answer
56 views

WGCNA: Problem with selecting soft threshold

I have 25 tumor samples with counts data. Initially, I filtered out low expressed genes and then converted counts to varianceStabilizingTransformation using ...
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1answer
76 views

Differential gene expression bias due to effect of an individual sample

I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various ...
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2answers
63 views

Is the quality score of fastq used somewhere besides trimming/fastqc?

In the fastq format every 4k+4-th line contains the positionwise qualityscore (ascii encoded): ...
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0answers
79 views

Error in my RNAseq analysis

I was following the RNAseq analysis tutorial online(https://combine-australia.github.io/RNAseq-R/06-rnaseq-day1.html) but am not obtaining the same variance values for each row in the logcounts matrix....
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16 views

include a glimma interface in a shiny app

I am trying to code a shiny app for RNA-Seq data analysis. I would like to include glimma interactive plots in it. However, in my current interface, clicking the action button ...
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1answer
24 views

mapping and de novo assembly of plant without reference genome

I'm studying on a plant that has no reference genome and only has one scaffold assembly and one gff3 annotation file. Can I create an index with the same assembly and gff3 in STAR and do the mapping? ...
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3answers
107 views

How to convert gff to gtf?

My annotation file is in .gff format. I would like to convert it to .gtf format or to know if there is a way to directly download the annotation file in .gtf format? I am working on sequences from ...
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0answers
18 views

What should I do when I have “#NAME?” in my Log Fold Change data to calculate Z-score? replace with zero or what?

I am trying to calculate the Z-score for several columns with Log fold change (LFC) data from RNA-seq expression values. But the mean for all columns, except one of them, is -inf and the standard ...
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1answer
100 views

how to interpret of “pvclust” dendrogram and finding height for cutting dendrogram?

I study on RNA-seq expression dataset about one cancer in TCGA. I downloaded FPKM dataset and removed batch effect by ComBat() function. Now, I used pvclust for ...
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20 views

How to detail the specific GO terms

How can I get more specific GO terms when using clusterProfiler? I got my Dotplot by: ...
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0answers
29 views

Model matrix design for limma-voom and batch effect correction

I've posted it on Bioconductor but didn't get a response, so I thought maybe I could get some help here (most likely the same audience but I'll try) I have multiple clusters in a 270 leukaemia sample ...
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1answer
26 views

Pre-filtering genes for Principal Component Analysis

I have a raw counts data-set of 20,502 genes and 137 samples. I want to find out Principal Components which best explain variation between samples in different stages of tumor. I am new to Machine ...
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2answers
37 views

about GO terms's name

Did anyone know whether the GO terms can include more detail information? Like I can get the DotPlot of the GO terms as below: The problem is that some of the genes, in the GO terms, is more about ...
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1answer
49 views

Where can I get ensembl Medaka genome for RNAseq

I am trying to map a RNA-seq dataset using ensembl genome for medaka fish. From here http://uswest.ensembl.org/Oryzias_latipes/Info/Index when I click on a ...
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1answer
53 views

RNASeq: Normalization, stabilization, gene length and rlog

I was thinking about the best method for normalization, which takes gene length into account (in order to compare genes)... Do you think I can do that? : - taking raw counts and dividing each gene by ...
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0answers
37 views

Should I use log2-CPM values (voom-limma) as input for my model?

We have created a model to integrate several OMICs data, but we realized that the maximum TPM values of RNA-Seq data were so big that had unexpected effects on our results. We hypothesized that this ...
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0answers
25 views

How to analyze expression of certain group of genes in certain types of cell?

I need to analyze expression of ~ 400 genes with a certain function in the embryonic stem cells. All these genes are combined into one database with RefSeq IDs. What is a recommended workflow to ...
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1answer
64 views

CIBERSORT runtime error eval failed

I was running CIBERSORT but caught this error right after it started permutation: ...
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1answer
96 views

Is re-normalization of RNAseq data recommended for analysis of gene subsets?

I downloaded an RNAseq dataset from TCGA database in 3 formats: 1) HTSeq counts; 2) FPKM; 3) FPQM-upper quartile normalized. The complete dataset contains ~60,000 genes. All of my analysis will focus ...
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1answer
60 views

RNAseq data analysis

I am currently doing a Differential Gene Expression of RNAseq data in Lung Adenocarcinoma using TCGAbiolinks. In the data Preprocessing step TCGAanalyze_Normalisation, I am confused as to which method ...
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1answer
47 views

How I deal with this expression set?

I have a gene expression raw counts like below for 16 patients ...
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0answers
30 views

How to segment genome into homozygote/heterozygote blocks based on RNA-Seq data

Given a RNA-Seq data of a progeny between two inbred lines (for example an F4 plant which was derived from two inbred lines (parents) and the F1 selfed 3 times; however this is probably not crucial ...
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1answer
40 views

Tree cut issue in WGCNA

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3answers
193 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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2answers
119 views

too long header for fasta file

I have a fasta file, like this: ...
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0answers
53 views

Selecting genes with more contribution from PCA

I have RNA-seq data in response to treatment vs non response; By machine learning I selected three principle components likely can predict the response based on the gene expression. Now I have ...
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1answer
79 views

Convert FPKM to RPKM

I have my 24 samples analyzed by Hisat2>Stringtie, from paired end sequenced data. I have got FPKM data and am planing to convert them to RPKM. to be able to compare my samples with another already ...
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1answer
19 views

How to map selected genes to Metabolic pathway Maps

I have a selected Arabidopsis Genome Initiative (AGI) list for RNA seq and proteomics data, how can I map them to metabolic pathway maps to vilualize(e.g. TCA cycle / FA / Photosynthesis) in KEGG or ...
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1answer
271 views

Changing the axis limits of ggplot objects

By EnhancedVolcano R package I have this plot with this code ...
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1answer
84 views

How I deal with this kind of gene expression comparision

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example tumor 1 in batch 1 and tumor 1 in batch 2 , ...
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2answers
258 views

TPM or rlog(CPM) for comparing expression?

I want to see the expression of a gene in a group of patient amongst the entire cohort using my RNA-Seq data. While I can do a differential expression analysis with limma or DESeq2, I want to see how ...

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