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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26 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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21 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
30 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
26 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
32 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
25 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
37 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
47 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. ...
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2answers
41 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
32 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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39 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
43 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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46 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
73 views

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

I have (gave me) Illumina fastq files 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 thanks
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2answers
26 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
60 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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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
28 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
35 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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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
30 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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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
41 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
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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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39 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
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What are the applications of DNA or RNA pattern matching?

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
35 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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20 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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56 views

Query on htseq count

This question has also been asked on Biostars I am trying to run htseq-count for carrying out rna-seq analysis for solanum tuberosum and i used the following command: ...
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1answer
38 views

What is the best way to map/align my reads on a given genome?

I am frequently using a ballgown package for my rnaseq analysis, but recently I have had a new task to have my reads mapped on two different genomes to understand the level of alignment between the ...
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1answer
69 views

What is a sensible number of gene/observations to explain PCA variance?

I am working with a set of RNASeq dataset. I have about 4000 observations (genes) on 20 samples and plotting a PCA I found the clustering doesn't vary much when I use different number of genes, but ...
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1answer
56 views

Question of Padj value (very high, infinite) got from dds

After I got dds from my count matrix by DESeq(), I use results() method to get Padj and log2 fold changes for volcano plotting, then I found some gene's Padj values are infinite and the P-value is ...
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2answers
58 views

How would we test for significant changes in expression when many of the normalised counts are clustered together?

I've been handed RNA-seq data with a lot of associated covariates. This data has been put through the DESeq2 package and as a result normalised the data. One of the transcripts of interest still has ...
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ConsensusClusterPlus for sample classification

Many large studies like TCGA use consensus clustering for transcriptional subtype identification. The most popular package for that seems to be ConsensusClusterPlus. If you want to classify an ...
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1answer
60 views

If a gene is expressed at a level of 1/1200 compared to the average gene, how is probability 50:50 that we have a read mapped to it?

I am reading a book about RNAseq analysis and it says "To calculate the probability that a read will map to a specific gene, we can assume an average gene size of 4000 nt (100 M nt divided by 25,...
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1answer
43 views

I am trying to run nab to create the pdb file but it isn`t working (AMBER)

Here is the input file for nab: molecule m; m = arna( "gcuucuucuucuucgc" ); putpdb( "lr16.pdb", m, "-wwpdb -nocid -tr"); I am trying to run nab to create the pdb file ...
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1answer
87 views

How to find the number of contigs produced? N50 length in base pairs? N90 length in base pairs?

I am having trouble with some underlying questions about my project. I have ran a Trinity tool to create a trinity.fasta file. To determine some underlying questions, I used the utility asm_stats to ...
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1answer
31 views

two aligners combined results

I would like to ask if someone uses two different aligners to produce count matrices and then run the same alogrithm for DEG analysis, would it make sense to find the intersection of the DEGs in order ...
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1answer
32 views

Gene expression Table to Expression Matrix converstion

I have an RNAseq gene expression file (Count data) as follows, I need to conduct a Differential gene expression analysis between Patients, for that, I need to have this file as a Matrix of Rows as ...
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36 views

Best way to align to find inserted sequence

We have some RNA from knock-in mice, there are two different sequences we're looking for. We have aligned to the mouse genome using STAR but the sequence isn't there which isn't too surprising What is ...
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2answers
59 views

Are sequencing reads written differently when inserts and indexes are sequenced seperately?

I was going over different sequencing library protocols and noticed that the adapter sequence can vary between protocols. In some adapter sequences the index sits between the primer binding site and ...
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1answer
172 views

DESEQ2:Error in rownames, what is the problem?

I am using DESEQ2 for RNAseq analysis but i do not understand why I get this error. ...
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1answer
63 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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4answers
120 views

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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3answers
49 views

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
49 views

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
40 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
42 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
48 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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