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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6 views

Running GSEA or comparable analysis with multiple variable interaction?

I am working with an RNA-seq dataset generated from a complex experimental design. Our main question is focused on the interaction between two variables, one (INJ) with two conditions (L,S) and the ...
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1answer
36 views

Setting Contrast for DESeq2 results

This question was also asked on BioStars I am trying to recreate this heatmap. How would I compare the four variables: Ly49+/- and MOG/MOGSP to get a single heatmap? Thank you for helping me through ...
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Advantages of paired-end sequencing compared to single end

One of the advantages of paired end sequencing over single end is that it doubles the amount of data. Another supposed advantage is that it leads to more accurate reads because if say Read 1 (see ...
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1answer
20 views

Pointing Cromwell to local EBS mounted storage on AWS Batch

I'm trying to run the WDL tasks from the GTEx RNA-Seq pipeline with Cromwell using AWS Batch as a backend. I store the STAR alignment index on an Elastic Block Store since my Cromwell server instance ...
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1answer
36 views

Error using htseq-count: Could not retrieve index file

I have a total RNAseq dataset that I aligned using STAR producing BAM files (sorted by coordinates). I am now trying to get counts for the lncRNA sequences using htseq-count, with the command: ...
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27 views

Which of the Transcriptome assembly method is best for identifying novel lncRNAs?

I'm working with human samples and I'm trying to identify novel lncRNAs from tumor samples of Prostate cancer. I'm using reference based transcriptome assembly with ...
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1answer
24 views

Analyzing transcripts for non-coding RNA

From a RNA seq experiment we look for the counts of transcripts of tRNA but we are also interested of other types of RNA we can find. Because of the protocol used for the library prep we can't analyse ...
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How to find siRNA off-targets from RNA-seq data?

I have an RNA-seq dataset from siRNAs targeting different components of a molecular pathway. the problem is that the siRNA ...
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1answer
35 views

the variation between treatments is less than the variation between replicates in RNA-seq data

I have a set of RNA-seq samples from targeting different proteins in a complex with siRNAs. However, the ...
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1answer
39 views

NO_COOR reads not in a single block at the end 0 -1

I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse "...
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1answer
47 views

TCGA: gene IDs not appearing and other concerns

I want to download RNA-seq datasets of 4 or 5 different types of cancer from the TCGA to investigate what is happening with my gene of interest. The problem is that I'm a physician and I don't know ...
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1answer
95 views

Removing Batch Effect in Heatmaps after Differential Gene Expression Analysis

I'm working on a dataset in which the first replicate of each group is one batch and the second replicate is in a second batch. After checking the PCA plot and ...
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1answer
60 views

Interpreting this plot from GSEA

I have RNA-seq for two groups of patients: Responders to chemotherapy (n=9) versus ...
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1answer
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Within and between sample count normalization

recently I came across a situation where RNASeq sample quality was expresses trough DESeq sizeFactor. So authors reported quality of their samples with respect to some publicly available datasets as ...
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2answers
34 views

why in 3' quantseq should we use reference genome and not transcriptome?

I am studying about RNA seq. I have found that in 3'prime rna seq (quantseq, tagseq) we should use a reference genome with good annotation (plus known UTRs). My question is why? Why could not ...
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1answer
90 views

why in RNA seq don't we only use reference transcriptome?

I would like to ask why in RNA seq analysis (alignment step) we use sometimes reference genome instead of reference transcriptome? thank you!
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2answers
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Recommended workflow for RNA long read Kallisto-like TPM estimation?

On short reads, Kallisto/Salmon is a standard workflow for measuring RNA transcript counts in TPM. However, when I tried to Google a similar workflow for long reads, it's not clear there is a ...
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What is a good rule of thumb for the threshold of noise versus signal for RPK in RNA seq?

I have RPK values (RNA seq) and I'm wondering what is a good rule of thumb for what is considered to be noise versus what is considered to be signal? I.e what should I choose as a threshold value for ...
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What is the best way to address the question of doublets and multiplets in a single cell RNA seq data set?

I have attached a histogram plot of the number of genes per cell in a single cell RNA seq data set of lung endothelial cells. I do not find a bimodal or multimodal distribution of the number of genes ...
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50 views

Differential Gene Expression with Replicates for some of the samples

[this question has also been posted on Biostars; some additional clarification from there has been copied into this question] I've been asked to analyse a set of samples in which their control sample ...
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32 views

a proper Design Matrix for several drug treatments with both control negative and control positive

I have a dataset of RNA-seq samples for testing different drugs on the presence of another drug. One of my samples is the normal cells with no drugs (control negative) and another is the cells with ...
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What is the best approach to classify a patient cohort by the expression (low, int, high) of a single gene of interest?

I am working with a dataset of nearly 100 patients. I performed Salmon quantification with genecodeV34 and imported the results with tximeta. I normalised the TPM salmon output with TPM (using edgeR, ...
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2answers
41 views

How to get transcriptome FASTA file for viruses for Kallisto pseudo-alignment?

I guess the title is self-explanatory. I'd like to find the reference transcriptome files for a few human viruses. My RNA-seq samples are from human tumor cells that were infected with viruses. The ...
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20 views

particular heatmap combinning to barplot for up and down-regulated genes

I have a list of genes (regulated up and down) by Deseq2 . I'd like to generate a heatmap of a particular combination for the barplot like in this graph : which I read in this paper: https://www....
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59 views

can I download DESeq2 in R 3.6.3 in Linux MInt?

I am trying to download DESeq2 in R 3.6.3. Is it possible? This is printed when I am trying: Warning in install.packages : package ‘DESeq2’ is not available (for R version 3.6.3) Thank you!
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3answers
93 views

RNAseq biological replicates not clustering in PCA plots

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster ...
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1answer
57 views

How to add cluster name to metadata in Seurat?

I'm working on a Seurat object and want to name the clusters according to 2 values alone (yes/no). So I want to add a new column to metadata and annotate the clusters (UMAP) with it. ...
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3answers
74 views

Download multiple fastq files using fastq-dump

I want to download the following fastq files at the same time in Salmon: - SRR10611214 - SRR10611215 - SRR10611215 - SRR10611216 - SRR10611217 Is there a way ...
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0answers
28 views

scRNA-Seq: Account for sequencing depth and gene length?

Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length. For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
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Removing Ribosomal RNA genes from Drosophila gene counts

Hi I have seen similar questions but I still need clarification on Removing ribosomal RNA genes from a gene counts matrix (Drosophila Melanogaster) Finding a fasta file with only ribosomal genes, I ...
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14 views

Detecting Transcript Isoforms with HISAT2/Stringtie RNAseq Output

I have the following Stringtie RNA-seq output files for several samples in the image below. For additional context, I have utilized as part of my RNA-seq workflow. HISAT2, samtools, and Stringtie ...
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4answers
47 views

How can I obtain FTP links to studies in ENA?

How can I programmatically obtain ftp links to RNA seq fastq files in ENA? Here's an example of a link that I would be interested in obtaining: ...
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Suggestion for a single-cell analysis: how to orchestrate a single cell analysis in order to infer the cell types?

I'm very new to scRNA-seq data so I'm sorry if I eventually posed a trifling question. I orchestrated a sc-cell experiment passing through several steps. First of all I eliminated all the genes that ...
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21 views

SCRAN encountered negative size factor estimates

I am running a public 10x dataset through SCONE in which one of the normalization techniques is from SCARN ...
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1answer
26 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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3answers
337 views

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
21 views

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
58 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
68 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
36 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
64 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
46 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
30 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
84 views

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
23 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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25 views

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
43 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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3answers
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
39 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
45 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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