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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14 views

RNA seq .counts.txt to bigwig conversion

I've downloaded neuronal RNA seq data from GEO. The files are in .counts.txt format. Could I convert them to bigwig? If so, how?
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How to reduce the occupied RAM when you are dealing with a very sparse matrix in a single-cell Experiment in R?

I'm dealing with a very large and sparse dataset and the first issues I met occurred when I tried to use quickCluster that reported me this error: ...
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1answer
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classifying samples by TCGA signature

I have some RNA-seq samples from multiple glioblastoma tumours that I'm now trying to classify according to a specific gene signature (from Verhaak et al., 2010) using R. The gene signature is ...
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0answers
35 views

How to calculate module-trait relationship when trait data is in binary format?

I have a dataset of 50 breast cancer samples. These samples are classified into four subtypes Lum A, Lum B, Her2 and Basal. I have been working with lncRNAs and protein-coding genes. To identify the ...
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2answers
48 views

How to create a list of differentially expressed (DE) genes after normalization with RUVSeq?

I am using edgeR to perform differential expression (DE) analysis on a set of RNA-seq data samples (2 controls; 8 treatments). To correct for batch effects, I am using RUVSeq. I am able to get a list ...
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2answers
30 views

Difference between isoforms and paralogs in transcriptoms?

I've assembled RNAseq data into a transcriptoms using Trinity. There's a option to keep only the longest isoforms for each transcripts and it lead me to wonder how it deals with duplicated genes (--&...
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1answer
48 views

Decontaminating RNA seqs for de novo transcriptome assembly and annotation of novel eukaryotes

I have raw paired-end RNA-seq reads for two novel eukaryotic species. Some background: the reads represent a copepod (arthropod) species each. The mRNA for each read set was obtained by extracting ...
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1answer
45 views

What are the state-of-the-art cell-type RNA-Seq deconvolution methods?

I would like to find the proportion of each cell-type in bulk RNA-Seq transcriptomics data. I am looking for some guidance on the following: What are the state-of-the-art methods? What are their ...
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1answer
28 views

RNA-Seq data transformation prior to sample correlation analysis

If I’m starting with Deseq2 normalized counts what are some preprocessing steps that I should apply to these data before estimating sample correlation using the cor function in R? For example, would ...
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2answers
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Problem with merge data while trying to convert gene names in R

I've been trying to code (in R) a way to convert gene accession numbers to gene names (from RNAseq data). I've looked at all the related questions and tried to modify my code such, but for some reason ...
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1answer
17 views

Comparing multiple treatments to multiple other treatments in edgeR for simple effects in a complex experimental design

I am working with a RNA-seq data set in maize that has a relatively complex design. There are two levels of treatment A (nitrogen fertilizer level in the field, high or low), two levels of treatment B ...
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Grabbing all Immune related genes with databases in R

I am having trouble grabbing specific pathway info using databases in R. I have RNAseq results and I want to remove immune related genes from the current list that I have. With a vector of gene ...
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1answer
33 views

about two group comparison of three group data in DESeq2 package

When we have three groups samples (A,B,C) with 25000 genes and the main interest is A vs B, should we limit samples only A and B for normalization to perform DEG analysis? Or better to include all ...
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29 views

Understanding the “relativeness” in RNA seq experiments

Let's say we have two samples, sample 1 and sample 2, and we're running an RNA seq experiment. Consider genes A and B for both. For simplicity, let's say that when we run the RNA seq experiment, we ...
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1answer
26 views

What is a good RNA seq normalization method that allows for across sample comparisons and between transcripts

What is a good RNA seq normalization method that allows for across sample comparisons, and allows between transcripts comparisons as well? I read that TMM for example allows across sample comparisons ...
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4answers
42 views

Files for paired end RNA sequencing

I am looking at the videos at a DIY Transcriptomics course and the speaker mentions that to run Kallisto for read alignment with paired end sequencing, one would enter the following: where the sample ...
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1answer
77 views

Which module to select for Pathway analysis based on Module trait correlation and pvalue?

I have a total of 35 tumor samples classified into 4 subtypes. Subtype A, B, C, and D. I have RNAseq data. I'm interested in identifying modules related to each subtype with co-expression network ...
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27 views

Regarding RNA seq data analysis and building coexpression network

I have some questions regarding RNA seq analysis if you can suggest anything it will help me a lot. I am currently normalizing RNA seq data for comparing genes expression within and between samples. ...
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29 views

Gffcompare issue: 0 reference transcripts loaded

I am trying to use gffcompare to compare my assembled transcriptome to a reference gtf that contains information about small open reading frames (sORFs). The reference gtf was obtained by processing ...
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2answers
40 views

Clarification about how mapping to a reference genome works in RNA seq

When mapping reads to a reference genome, how is it possible to tell which part of the genome the read is referencing if the genome possibly has the same repeated sequences in different areas? What I ...
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0answers
16 views

Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data

I am following the tutorial here to learn how to process raw RNA-seq data and get gene read counts. I've had no issues until I got to step 3.9, "Assignment of RNA-Seq Reads to Genes." Here, the ...
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2answers
31 views

How to tell if library generation for RNA-seq experiments are stranded vs not stranded

I would like to use RNA-seq data from the NIH GEO database. How can I tell if library generation for the RNA-seq experiments are stranded vs not stranded?
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1answer
36 views

WGCNA co-expression network analysis with less than 20 samples

I have two cancer subtypes data. Subtype A is 14 samples and Subtype B is 23 samples. I'm interested in identifying the functions of some LncRNAs in the Subtype A group. For this I'm using all protein-...
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20 views

apply TMM on counts imported from salmon using Tximeta

I used Tximeta to import a summarisedExperiment from the salmon output (used with genocide transcriptome v34). I need to produce 4 matrix of counts: - tx counts in TPM - gene counts in TPM - tx counts ...
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14 views

extracted counts matrix from Tximeta generalized experiment with no linked transcriptome are normalized?

I used the Tximeta vignette to construct a summarised experiment from quant files (salmon, index prepared with NCBI transcriptome). 1)Tximeta return me a non-ranged se (which I am not sure about the ...
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7 views

Cuffdiff multiple comparison

I have four groups G1,G2,G3 and G4 all in triplicates, I want to make the comparison of differential expression between the four groups together used cuffdiff following: ...
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1answer
38 views

How to incorportate RIN values as covariate in the design matrix?

I have been following the last DESeq2 pipeline to perform an RNAseq analysis with a dataset with low rin samples in the experimental (or treated) and high rin on the control ones. I read a paper in ...
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7 views

Does “Wrapping up the results” in ballgown means that ballgown object is created correctly?

I am trying to apply a Pertea et al. procedure to my RNASeq samples for which I have created a ballgown folder as explained in the article. When I am trying to prepare my phenodata file for every ...
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1answer
38 views

How to understand and analyse RNA-seq data (for a beginner)?

I am trying to understand expression of a certain protein across Pseudomonas species. I downloaded an SRA file from NCBI and converted it to a fastaq file. I am not able to understand how to interpret ...
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1answer
52 views

Should biological replicates be the most similar pairs in an RNAseq experiment?

The attached figure (from deseq2) shows the sample to sample distances of a RNAseq experiment with 4 conditions (A,B,C,D) at 2 time points (0h,4h) with 2 biological replicates. I am a little bit ...
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2answers
29 views

cellranger mkfastq with full path to --id flag

I have always used cellranger mkfastq to demultiplex 10x genomics runs manually, though recently the commands to do so have been incorporated into a script that ...
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0answers
41 views

PAM50 gene expression classification

I'm looking at running the PAM50 classifier on RNA-Seq from 138 breast cancer samples. However, the R package (genefu) that's useful for this does not have a ...
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1answer
32 views

Why this ballgown error (first column of pData does not match the names of the folders) comes out

I am trying to create a ballgown object, and I would like to set a phenodata information. For that I do phenodata <- read.csv('l002.csv') and my l002.csv ...
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0answers
29 views

vst() from DESeq2 vs voom() from limma

I have used both to transform my leukaemia RNA-Seq data for subsequent hierarchical clustering. The result is quite different. Some subtypes of leukaemia only form a cluster (or at least sit closer) ...
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0answers
21 views

How to convert Illumina IDs to Gene Names?

I am new to the area of bioinformatics, so apologies if this is too obvious of a query. I need to analyze the RNA Seq data from GSE98455. The dataset is of the following format: ...
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28 views

Is there a single cell RNAseq equivalent of GTEx or TCGA? [closed]

Or do I need to find individual studies and obtain data the long way!
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1answer
55 views

How can I bypass 'out of memory' problem using a HISAT2 for human genome indexing?

I am trying to perform a HISAT2 indexing using a GCF_000001405.39_GRCh38.p13_genomic.gtf and ...
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1answer
66 views

How to downsample some of the samples in RNA-seq data?

I have 40 samples and these are into two groups. I would like to perform a differential analysis between two groups. The library size of the samples is very low. But there are two samples (GroupA_12 ...
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17 views

reactomePA dataset

I tried to create my geneList according to this r code: setwd("C:/cygwin64/home/DIANGO/EXCELL/") d = read.csv("PA_down_id.csv",sep = " ", header = F) head(d) ...
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1answer
94 views

Downloading SRA Files from AWS

I want to download the original BAM files that the authors had uploaded to SRA. Normally, I would just use sam-dump, but the files are having issues that seem ...
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1answer
34 views

Database of RNA-Seq and SNP genotyping data

I need to obtain publicly available RNA-seq + SNP genotyping data for a cohort - ideally one where the RNA-seq data has been sequenced from brain tissue. I have found CMC and GTEx to be sufficient ...
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0answers
12 views

Pantherdb Export

After a gene enrichment analysis in the panther database I would like to export my graphic data (barplot and Pielot). Then I would be happy with a suggestion or advice from which will allow me to ...
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1answer
35 views

How to download RNAseq gene expression data from GTEx

I would like to download RNAseq gene expression data from GTEx from the liver only. What is the best way to do this? I have tried a few things. I downloaded several files from the GTEx website (https:...
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2answers
56 views

What RNA-Seq expression value would be closest to Microarray equivalent?

I know this question may seem strange. I'm using Spearman correlation between gene expression profiles for various reasons (I won't go into details here). As a result, I often compare RNA-Seq and ...
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1answer
89 views

Differential Expression

I have genes differentially expressed between two groups (case and control). I would like to annotate them by classifying them according to their biological functions. I work on parasites of ...
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2answers
893 views

How can I classify the 3 clades(S, G, V) of the coronavirus without using protein data?

On GISAID they classified the coronavirus using 4 clades(S, G, V, Other). I downloaded around 1,000 complete genomes of the coronavirus from GISAID and I would like to classify each one as belonging ...
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1answer
38 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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24 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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1answer
19 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
30 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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