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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20 views

Parallelizing microRNA targets

I am trying to look for miRNA targets using a file called Conservedfamily.txt from the Zebrafish target scan fish website. I have written a python program to ...
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1answer
37 views

DESeq2. Which is better: using the altHypothesis argument in the results function or manually filtering for your desired P-value and Log2FoldChange?

In DESeq2 you can identify your differentially expressed (DE) genes using the results function. I noticed people in my lab using the results() function with the minimum number of arguments supplied (...
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3answers
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The confusion of using TPM (transcripts per million)

It is shown that TPM values are not suitable for DEG analysis but good for within-sample comparison since TPM normalized the gene length. My question is first: if TPM is not suitable for across ...
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0answers
7 views

Taiji doesn't run Pagerank

I'm trying to use Taiji to run a pagerank on our combined bulk RNAseq and ATACseq dataset. Here's how we run it: taiji run --config config.yml -n 10 Here's ...
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1answer
39 views

how to get normcounts for singlecellexperiment object?

I need to perform differential expression analysis using the scDD package from R, but I am not able to since I miss the normcounts assay in my SCE object (of course in the example they show, the assay ...
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1answer
40 views

Understanding “Measurement of mRNA abundance using RNA-seq data” by Wagner et al 2012

I'm trying to understand why RPKM is not an appropriate way to normalize for RNA seq (I understand the general idea, but I'd like to gain a deeper understanding). So I'm reading the original paper by ...
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0answers
33 views

Correcting for batch effect in bulk RNA seq datasets

I have samples from many different bulk RNA seq studies that exhibit an evident batch effect (samples from different labs cluster together regardless of treatment). I've found a lot of papers (here, ...
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1answer
12 views

Combining read counts from three separate GEO studies

I want to do differential expression analysis with DESEQ2. I have three read counts files downloaded from GEO (small RNAseq based) where the number of miRNAs and id is nearly the same. These studies ...
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1answer
45 views

Batch effect correction using a subset of samples - using DE genes

I have some RNA-seq data with two very obvious batches as you can see in the PCA: The samples of interest (A - H) are from tumor tissues. In addition, there are data for two cell lines I and J. The ...
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12 views

Installation of software Capmirseq

I am trying to install a software called Capmirseq. After installing all relevant modules I followed the installation instruction code as follows ./install.sh -p /path/to/software. Which is expected ...
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1answer
37 views

RNA_Seq data aligned used uniquely or multi mapped reads impact on result interpretation

I have some transcriptomic (Whole) sequencing data that I should analyse. I would like to do raw data alignment to a reference genome taking into account the multi mapped reads and uniquely mapped ...
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0answers
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Ccle miRna data units

Does anybody know what units the mirna expression data on ccle is in? I saw the pipeline in the original paper but it doesn't mention units. Original paper: https://www.nature.com/articles/s41586-019-...
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How to create a DESeqDataSet and define experiment design before varianceStabilizingTransformation?

I have an RNAseq count matrix consisting of 2 groups (high, low) with 6 timepoints per group (T1,T2,...,T6) and 3 replicates per timepoint (rep1, rep2, rep3). So a 2-factor design with 36 samples in ...
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1answer
43 views

How does FDRtool work?

I have a question about using FDRtool. In the below code (on RNA seq data whose p values were acquired using Deseq2), the FDRtool was first used and thereafter p.adjust using the benjamini hochberg ...
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2answers
143 views

Calculating most abundant transcript from RNA-Seq data

vcf2maf uses VEP to annotate variants, and I believe selects the default Ensembl transcript to use for annotation. Sometimes the ...
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1answer
44 views

Get Salmon mapping/alignment summary

With HISAT2, after the alignment of fastq files you get an alignment summary like this: ...
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1answer
45 views

Base abbreviations other than ACTGU in sequence file

Short question: where can I find what F and J and < and ...
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1answer
54 views

What is the meaning of split read?

I want to use rna seq data to later perform functional tests on fusion genes. so before that I need to filter the "best results" (of rnaseq) for deciding which candidates I actually want to ...
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1answer
75 views

What are common ways to calculate gene length for TPM calculations? [duplicate]

I am aware of this similar question. But the accepted answer there answers how to calculate TPM given a mapping from gene name to gene length. My question is, given an annotation file of genes (for ...
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2answers
38 views

RNAseq DE comparison with samples of different read length [duplicate]

I have RNA samples but with different read lengths (Eg, HiSeq 2x125 and NovaSeq 2x150bp data). I would like to do DE analysis on these samples. What do you recommend? Do you recommend to trim the ...
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5answers
108 views

Normalize RNA seq data from multiple runs for expression analysis

I have RNA samples sequenced with TruSeq Stranded Total RNA kit protocol in Illumina HiSeq (2x125bp) and NovaSeq platforms (2x150bp) - almost 100 samples altogether. I have to use the samples data for ...
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1answer
45 views

correlation coefficient versus DEGs analysis: what's the best approach for low expressed genes?

I have 6 folders. Each one contains 7 datasets of a specific type of cancer (RNA-Seq) and 7 datasets of normal tissue (healthy control for that type of cancer). A total of 84 datasets. I want to ...
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0answers
26 views

Data format for pathway based clustering of samples

I came across this paper as one of the examples from this paper, this one Figure 2. Host Protein Alterations in Infected iAT2s ...
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1answer
42 views

Differential gene expression analysis of time series with replicates

I have a dataset that has two groups, perturbed vs control. Each group has 3 replicates. Each replicate has 8 time points. How do we do Differential gene expression analysis to find significantly ...
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1answer
241 views

Why did expression based subtypng of breast cancer gain much more acceptance than others

This is may not be entirely technical question but rather a academic question. But the technique behind the application is within the scope of bioinformatics. So I would try to ask here that: In each ...
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1answer
41 views

Toptable error, wont recognize condition

I am getting a rather strange error from topTable. When I run my code I get Error in fit$coefficients[, coef] : subscript out of bounds from topTable as if it is ...
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1answer
40 views

Challenging benchmarks for supervised learning on sparse scRNA-seq data

One challenging aspect of modeling scRNA-seq data is data sparsity, that is, scRNA-seq measurements typically suffer from large fractions of observed zeros (i.e. dropouts), where a given gene in a ...
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1answer
35 views

Demultiplexing FASTQ file without index information

I am trying to understand how data that was uploaded to SRA (https://www.ncbi.nlm.nih.gov/sra?LinkName=biosample_sra&from_uid=4510743) can be analyzed with the assumption that the FASTQ file ...
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1answer
61 views

RNASeq analysis using featureCount and EdgeR

I am using a pipeline (bam -> featurecount-> EdgeR) to do some RNASeq analysis of several groups and sub-groups. For example, I have the following dataset with two types (T1 and T2) and T1 has ...
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2answers
74 views

Un-normalize DESeq2 counts

I obtained a matrix of RNA-seq count data that has been normalized by DESeq2's median of ratio method. I know that DESeq2 wants to take in un-normalized counts, but I do not have access to those data. ...
2
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2answers
67 views

Calculate genome coverage and depth from alignment

I have a .bam alignment file and a genome reference .fasta file. I am looking for a easy to use tool (that I can reference in a publication) to calculate the percentage coverage of the reference by ...
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1answer
42 views

Common gene naming conventions and how to convert between them

I have two questions, one is a direct question about some RNA expression data. The other is more broad, but motivated by the first. I have downloaded an expression set for a class project and I am ...
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1answer
71 views

Doubt about using TPM for statistics

We designed an experiment to explore the potential role of carbon dioxide on algae physiology using RNA-Seq. We analyse the differential gene expression using DESeq2 but now we are interested into ...
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1answer
51 views

Determining what RNAseq data is filtered on volcano plot

I am using RNA seq data to analyze genes via a volcano plot comparing differential gene expression of bacteria with and without antibiotic in R. After having created my plot, I am unsure why some of ...
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0answers
64 views

How to use combat in order to remove batch effects?

I have RNA seq data and I need to use combat to remove the batch effects. Somehow when I run it, it isnt actually doing anything. The code: ...
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2answers
106 views

Fastq: how can I check if they are from DNA or RNAseq data?

I have (gave me) Illumina fastq files, which I want to use for variant calling, and I do not know if they are DNAseq or RNAseq data. How can I check this? I do not have any report or who to ask. Many ...
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2answers
35 views

Bulk RNA-Seq Read Length Normalization across different samples

I have 20 samples out of which 14 are 100 bp in length and 6 are 150 bp. Is there a way to normalize the read length for cross-sample differential expression comparison? What would be the best way to ...
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3answers
66 views

Explanation for RNA-seq samples not clustering in PCA as expected

A colleague is analysing RNA-seq data - the study design is 2 treatments, 3 replicates, 3 tissues. In their PCA plot the samples clustered neatly by tissue. Except for two samples - two tissue samples ...
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0answers
20 views

Trouble aligning next generation sequencing data to reference genomes using QuasR package in Bioconductor. Cannot import .txt

I'm trying to check the quality of my paired end read sequencing data. I am following this pipeline (https://f1000research.com/articles/4-1062#ref-21) which uses QuasR in the first step. My list of ...
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1answer
31 views

Kallisto psudoaligner quant not giving me expected output

I am trying to run this kallisto (latest) quant and all I get are abundance and json files. ...
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1answer
41 views

Clustering RNAseq fold-change data

I have a dataset that looks something like this Treatment1 Treatment 2 Control Sample1 3.23. 0.87. 1 Sample2 1.71. 1. 1 Sample3 2.88. 5.65 1 Sample4 0.77. 1.34 1 The numbers describe a fold ...
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0answers
40 views

How do you determine the strandedness of a RNAseq dataset? [duplicate]

How do you verify if a RNAseq dataset is unstranded, stranded or revesely stranded from a fastq file?
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1answer
32 views

Multi Omics comparison RNA seq : Condition specific gene signature

The authors of an article are comparing different cell type and the organ type and they show the ontology enrichment of the RNA-seq: Cell-type-specific and organ-specific marker genes were identified ...
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0answers
31 views

workflow for processed RNAseq data analysis in R [closed]

I'm learning to use R in data analysis. I'm getting fluent in baseR and the tidyverse, but thus far only dealt with medium throughput plate-based experiments. I am currently trying to learn how to ...
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1answer
72 views

RNA_Seq Analysis in R, propmapped function issue

I'm currently trying to learn RNA-seq data analysis and differential expression in R. I've been using the Combine lab tutorial. Everything was running well until the tutorial asked to use the ...
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0answers
18 views

TCGA dataset: different accession IDs mapped to same location?

I'm currently working on TCGA miRNA dataset. After constructing a reads matrix, I'm trying to find the isoform sequence. In my data, I have the genomic location (isoform_coords). I found that entries ...
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0answers
42 views

ATAC and RNA seq Up regulated region gene and scatter

I m not sure if I'm going the right way or not but when the gene and region and both upregulated in RNA seq and ATAC seq data I should be seeing a positive correlation but it seems other wise. Am i ...
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4answers
130 views

What are the applications of DNA or RNA pattern matching? [closed]

I'm assuming we don't do pattern matching in DNA or RNA for the fun of it. So I'd like to know what are the applications of pattern matching or where does it fit in in larger applications? I'm a ...
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1answer
39 views

Clarification regarding (deepTools) bamCoverage output

Im using bamCoverage on a file and ive set the bin size to 1 but also normalizing using RPKM for defined regions using a bedgraph file. The output from bamCoverage however is in binned segments each ...
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0answers
21 views

How to control/normalize for number of reads when calling SNPs using RNA-Seq?

I used the GATK pipeline to call SNPs on males and females using RNA-Seq data. But the males have a higher read count (~43-46M reads) than the females (~40-42M reads). This causes SNP counts to be ...

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