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

less than 80% of your list has mapped to your chosen identifier type

I am trying to do GO analysis. However, when I run DAVID, I am getting this error: You are either not sure which identifier type your list contains, or less than 80% of your list has mapped to your ...
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I have really skewed RNA-seq data, what's the best way to normalise it? Preferably in R!

My data frame compares the RNA-seq reads from many genes in different tissues. The reads look as follows. I tried using log to make it better but still looks pretty skewed. Are there any better ...
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Where can I find Single Cell Data with Location “Coordinates”?

Does single cell data typically have the following meta-data: the "coordinates" (e.g. on a tissue, adjacent tissues) saying where each cell in the sample was located relative to other cells? ...
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1answer
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Why use “robust” FPKMs?

Both DESeq2 and edgeR have an FPKM/RPKM function that by default uses normalized library sizes ("robust" option in DESeq2). FPKMs have their own issues, but I thought the main benefit was to ...
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1answer
23 views

Table error when combining counts columns

I have these files that I want to make table of (https://github.com/learnseq/RNAseqfiles01.git), the table that I want (since all the files have the same ID column),, I want merge the ID column with ...
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1answer
40 views

Splitting data table up based on separate file of gene names

I am very new so bare with me. I have a file called data (.csv that I turned into a data table) with 2 columns (geneid, normalized counts) and about 9000 rows. I have a second file called Xgenes of ...
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1answer
31 views

How to find adapter sequence

I have this GSE dataset ( GSE104279 ) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104279) I want to run cutadapt, but how would I find the adapter sequence so I can run cutadapt?
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Error in cor(exprs(gse), use = “c”) : no complete element pairs

I'm trying to plot a heat map for a "GSE133399", I retrieved the GSE using GEOquery package and then assigned to an object and then tried to plot a heatmap, but I was not successful, I used ...
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1answer
48 views

PacBio long-reads impact in transcriptome de novo assembly?

We are strongly interested in assembly a good transcriptome of reference for a non-model organism and build a local database. We have sequenced the same individual with Illumina (150 millions of pair-...
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1answer
28 views

Power law distribution alpha values

I did a Pearson co-expression analysis for generating networks for my tissue-specific (chondrocytes) RNA-seq data. I used R package poweRlaw to check for the power-law distribution. We got the ...
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normalizing transcript-level expression data

Generally, RNA-seq analysis tools like edgeR or DESeq2 work on gene-level data. For differential expression (or differential transcript usage), transcript-level data has its own caveats. However, for ...
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1answer
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smallest length of reads in 3'quantseq?

I am studying about RNA seq, and especially about 3'Quantseq(tagseq, 3primeseq). I wonder if there is a cutoff for the reads length. By this I mean that given that 3'quantseq targets the end of the ...
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1answer
35 views

data visualization RNAseq : scaling data for PCA and cluster dendogram

I have count data from a RNAseq experiment (2 samples are from normal cells and 3 samples are cells with a disease), and the data is already standardized by trimmed mean of M values (TMM). I want to ...
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Does rRNA depletion protocol give higher number of mapped reads in Intronic regions?

Recently, I have downloaded a publicly available dataset, which are 350 tumor samples. I see the following information from the published paper. They used Ribo Zero Gold and rRNA was depleted. Strand ...
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1answer
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if I know the number of sequencing circles can I give this information to DESeq2?

I am trying to understand library normalization in DESeq2. I would like to ask the following: I know that some samples have been run 15 cycles and some others 20, can I give this information to DESeq2,...
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Identifying nested/overlapping reading frames in nucleotide sequences

I'll start by saying I'm a total stranger to this field, so I'd love to be corrected if I misunderstood something along the way. I'm writing a python code to identify all the reading frames in a ...
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2answers
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Mapping statistics from bam file using bbtools and sambamba

Below are the statistics for RNA-seq mapped and unmapped paired-end reads to rice genome using reformat.sh from bbtools on bam files. It gives 77% mapped and 5% unmapped, what about the remaining 18% ...
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1answer
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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
42 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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2answers
89 views

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
23 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
145 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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34 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
38 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
45 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
53 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
130 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
66 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
49 views

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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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
92 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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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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34 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
55 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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88 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
109 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
111 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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117 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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31 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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28 views

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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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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3answers
51 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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