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

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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23 views

workflow for processed RNAseq data analysis in R

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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25 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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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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36 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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1answer
25 views

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
25 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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19 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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45 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
31 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
50 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
47 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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55 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
58 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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38 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
61 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
28 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
54 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
66 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
30 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
114 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
45 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
48 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
33 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
41 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
41 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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1answer
16 views

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
53 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
36 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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22 views

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
21 views

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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2answers
100 views

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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30 views

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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2answers
50 views

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
44 views

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
27 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
51 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
184 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
37 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
419 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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20 views

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
42 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
75 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
60 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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