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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Is loss/gain of function reflected in RNA-seq transcript counts?
Do LoF/GoF transcripts count toward the RNA-seq TPM count? Or would these LoF/GoF transcripts only be detected by isoform quantification?
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2
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75
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How should I interpret DGE results if only one HLA-A gene shows up as significant but not the others?
I have done a DGE recently and have been looking at the DGE list. One of the genes is HLA-A. However, when I dug deeper I realised there are hundreds of HLA-A genes with unique ENSEMBL number (of ...
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Expression analysis of miRNAs with normal RNA-seq data without small RNA-seq data
I am looking to perform expression analysis of miRNAs with normal RNA-seq data lacking small RNA-seq data?
Which path should I choose for known miRNAs and unknown new miRNAs?
Data set: rna-seq data ...
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21
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Genome-guided transcriptome reconstruction: should I filter my reference annotation by transcript_support_level or other tags?
Is there really any advantage to filtering the GTF annotation for transcriptome reconstruction (or, for example, for pseudo-alignment quantification)? Are there any downsides (i.e. reasons why I ...
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40
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GSEA. Subsetting Gene Ontology, Biological Processes, by gene set families of interest
I have run GSEA - on Gene Ontology , Biological Processes (GO and BP hereafter) - on some Mouse bulk RNA-seq data, and am currently in the process of going through the results. To do this, I have used ...
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Can multiplexing in Sequel II SMRTcells reduce the coverage?
I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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37
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How can Iso-Seq reverse transcriptase artifacts be avoided?
My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
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31
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Transcriptome analysis
I am trying to assemble reads belonging to two different readlength. Is it a valid way since I am looking for common genes among the species I am assembling.
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68
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RNASeq NMF clustering using what kind of expression unit?
I would need some help regarding certain points in a RNASeq gene expression analysis (sorry if there are stupid questions, its my first project and i have no capable supervision). My workflow is the ...
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2
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60
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Tool for cross referencing a newly found MOTIF to database of known MOTIFS
I need a tool or a function I can use in my code (R, or Python) that I can cross reference a MOTIF against known MOTIFS, a function that will take as input a MOTIF (a probability weight matrix, PWM ...
- 301
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53
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How to use the contrast function in ebayes for making different comparisons
The design matrix i have is this I would like to know how to use they way its done in deseq2 where we can use the contrast function to make particular comparison
The code im running to make ...
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79
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How to assess quality of WGCNA module identification in blockwisemodules()
I'm exploring WGCNA for bulkRNA sequencing analysis with human subjects. I have healthy, myocarditis, and heart failure patients (which can be further broken into ischemic or nonischemic)
I have been ...
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35
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Gene/protein expression specific to a group in omics
I am wondering what is the significance of finding a particular protein specific to a disease or control group? when we detect 1000s of proteins in a proteomics experiment, how can one be sure that ...
2
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4
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165
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nested samples in design DEseq2
I'm running a DEG analysis with Deseq2 by specifing the following design that includes 12 samples from 6 pts in 4 different conditions:
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220
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How to adjust by multiple variables using ComBat-Seq?
I am trying to adjust my RNA data using ComBat-Seq (from sva R package) since I realised that there are 3 batches that I need ...
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2
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262
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PWM (4xM matrices) to Sequence Logo Visualizations in MATLAB or Python
Is there a MATLAB, or else Python (not R) tool for visualizing sequence Logos from probability weight matrices (PWMs)?
This seems like a basic and simple tool to implement, yet just above the ...
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How to identify genomic regions / peaks associated with enhancers (TF binding sites)? Is there a tool or a formal recipe?
I am attempting to identify DNA sequences/regions associated with enhancers in my NGS sequencing data. How can I identify (from peak files/counts for chip-seq, atac etc.) genomics regions/peaks ...
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2
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162
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How to identify which isoforms of a gene are actually expressed in my data?
I am new to bioinformatics and have been assigned the task of discovering which isoforms of a certain protein-coding gene are present in the RNA-seq data that I have. There are several isoforms of ...
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120
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plotting gene expression after EdgeR DE analysis using RUVg (RUVseq) covariates
I have used the empirical RUVg method (from RUVseq) to estimate the unwanted variation of my dataset (consisting of several public datasets analysed together, with controls and case samples in ...
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129
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Extracting clusters, hierarchical clustering - bulk RNASeq
I have been attempting to extract particular clusters, given by hierarchical clustering outputs from the pheatmap function (in R).
Please find the ...
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217
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Tutorial for the WGCNA: changes in heatmap colours
I am trying to reproduce the results of the R tutorial of the WGCNA package.
In section I number 5, when generating the heatmap it is quite similar to the one provided by the pdf but the colours of ...
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Find enriched processes in modules obtained by WGCNA
I am doing a gene enrichment of different clusters obtained by WGCNA, but there are some of them in which I don't get enriched processes, is there any way to find processes to these modules, for ...
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92
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Can I Incorporate svaseq() into GSEA/GSVA analysis?
I understand GSEA/GSVA can take microarray-like expression matrix such as the output of voom() or vst(). However, I have a question on how we can also use svaseq() variables, correct the output from ...
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Different results of spearman correlation between TPM and FPKM
TPM and FPKM of RNA-Seq data form GDC TCGA calculated based STAR were retrieved, respectively. The correlation between a specific gene, e.g. HIF1A, and other genes were calculated based on TPM and ...
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DeconRNASeq: Extract gene names from returned mixing proportions
This question was also asked on Biostars
I am using the Bioconductor package "DeconRNASeq" to perform tissue deconvolution. Let's say I run the following code (this is from the manual):
<...
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1
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52
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statistical test and visualization of genes present in an experiment
I have a list of genes (n = 120) that are involved in breast cancer. And I have a list of differentially expressed genes (n = 70) that are present in the breast cancer gene list. My question is: Are ...
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3
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75
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beginner RNA-seq Replicate papers
have a good R and statistical analysis background (also with machine learning). in addition, I'm a fresh biotechnology grad. I would like to try to replicate some Rna-seq analysis with R papers (with ...
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77
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How can I get data of single cell RNA sequence with raw count?
I am using dataset GSE85241, but I can't find the read counts of the dataset. It only provides with RPKM values.
How can I find the read counts of a dataset?
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Is this RNA seq data good based on the MDS plot
I am analyzing some RNA sequencing data from a collaborator where the effect of some ligands on the transcriptome is being looked at. I am using DESeq2 for my analysis. I am looking to compare each of ...
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74
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Questions about CreateGeneActivityMatrix function in Seurat
Seurat V3 has a function CreateGeneActivityMatrix, which can convert ATAC peaks to gene-level's matrix.
Here is the prototype:
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293
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How to extract reads that map exclusively to a single site with 1 or zero mismatches from BAM files
I generated BAM files (sorted by coordinates) by aligning human RNA reads against the human reference genome using BWA MEM and ...
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how to create "sample file" for the qAlign() function after trimming the reads in R
I'm an absolute beginner trying to solve this question "Align the trimmed and untrimmed reads using QuasR and plot alignment statistics, did the trimming improve alignments?"
I did trim the ...
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180
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What is the difference between Normalized Expression in EdgeR vs DESeq2?
I am trying to access the normalized expression in both edgeR and DESeq2, yet the results are different. Does anyone know why?
How to get normalized expression using edgeR:
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baseMean threshold
I have an RNA-seq dataset and I am using DEseq2 to find differentially expressed genes between the two groups. I used pre-filtering to remove any genes that have ...
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103
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Error in DESeq2 analysis with dimnames
I'm getting the following error in a DESeq2 analysis and I can't seem to figure out the issue:
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2k
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DimPlot - How to highlight cells with identity colors?
I made this wrapper for DimPlot:
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2
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283
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How will Seurat handle pre-normalized and pre-scaled data?
I want to use data from the following dataset:
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84371
The data has been TPM normalized, which is not ideal for clustering but I have to work with ...
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RNAseq small sample size in control group
I am doing an RNAseq experiment with affected (n=6) and control groups (n=6) in cow. However it turned out that 4 of my control samples have very low quality so I have to discard them.
My problem is ...
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68
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Pulling out genes in a scatterplot
I'm comparing gene expression among 2 different datasets (in vivo and in vitro) I have made a heatmap showing the correlation for each and then plotted the data frame to create a scatterplot. Now I ...
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Target genes for piRNA
Where I can find a database or tool to give me the target genes of PIWI (piRNAs) in human?
I found one but works for worm like <...
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RNAseq SNP discovery: deciding upon filters and dealing with allele expression bias
I am working with non-model plant RNA samples which we have been deep sequenced and analysed using STAR aligner under default parameters.
Aim We would like to conduct SNP discovery of these samples.
...
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Workflow for identification of new splicing variants from RNA-seq data?
I'd like to perform a search for possibly unidentified splicing variants of a specific protein in A. Thaliana. I do not have my own RNA-seq data. It has been suggested to me to use this workflow:
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130
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Trinity de novo assembly not completing
I'm trying to assemble some transcriptomes using Trinity, but am having issues getting Trinity to finish. I've been trying to get this to work for weeks, but am hitting a wall and would very much ...
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Low mapping reads for RNA sequence
I am new to RNA sequencing analysis. I have RNA samples for bacteria for different treatment. I did contaminant filtration, remove adapters and checked fastQC report: attached below for one sample (...
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subsetting more than one ccle sample
I'm working with the CCLE dataset and I'm trying to subset the data to just 3 cell lines of interest but not sure how to it.
Currently, I have been subsetting one by one but is there an easier way?
<...
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Why is bulk RNA sequencing reflecting AVERAGE expression but not TOTAL expression of all cells?
When I am reading papers that compares bulk RNA sequencing and single-cell RNA sequencing, we often see papers describe bulk RNA seq measures the average cell expression.
For example, in this paper ...
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540
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Plotting a gene in Seurat
I saw in the extensive Seurat documentation for Dimplot (dimensional reduction plot), here, you can plot a gene by specifying it with group.by = "gene" but this does not work in practice.
<...
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2
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160
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Comparing differential expression across samples - is batch effect correction needed?
I have a bulk RNA-seq dataset made up of control and treatment conditions for a range of cell lines. This dataset was generated in two batches, such that the cell lines are split between batches but ...
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1
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102
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Normalization methods for single cell RNA sequencing that take read count into account
I have two RNA-seq datasets. One was sequenced at an average read count of 1.5 million per cell the other at 43K average reads per cell. For the first I also have the meta data from reads alligned ...
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