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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15
votes
2answers
2k views

How can we distinguish between true zero and dropout-zero counts in single-cell RNA-seq?

In single-cell RNA-seq data we have an inflated number of 0 (or near-zero) counts due to low mRNA capture rate and other inefficiencies. How can we decide which genes are 0 due to gene dropout (lack ...
15
votes
4answers
4k views

How to compute RPKM in R?

I have the following data of fragment counts for each gene in 16 samples: ...
11
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4answers
2k views

What methods are available to find a cutoff value for non-expressed genes in RNA-seq?

I have a gene expression count matrix produced from bulk RNA-seq data. I'd like to find genes that were not expressed in a group of samples and were expressed in another group. The problem of course ...
18
votes
3answers
3k views

How exactly is “effective length” used in FPKM calculated?

According to this famous blog post, the effective transcript length is: $\tilde{l}_i = l_i - \mu$ where $l_i$ is the length of transcript and $\mu$ is the average fragment length. However, typically ...
7
votes
2answers
347 views

differential gene expression complex design no replicates

We have an experimental design as seen below Where we administered drug at 0 min for each mouse genotype and took them down at given below intervals. wt and ko mouse models were administered only ...
2
votes
0answers
389 views

Filter out PCA outliers automatically

I am new to bioinformatics and PCA. What I am trying to do is to remove bad cells from a dataset that was obtained with scRNA-seq for ...
8
votes
1answer
2k views

How to apply upperquartile normalization on RSEM expected counts?

I see that TCGA RNASeq V2 RSEM data is normalized with upper-quartile normalization. After doing Quantification with RSEM with the samples I have, I got "genes.results" as output which has gene id, ...
13
votes
2answers
2k views

Normalization methods with RNA-Seq ERCC spike in?

ERCC spike-in is a set of synthetic controls developed for RNA-Seq. I'm interested in using it to normalize my RNA-Seq samples. In particular, I'd like to use the spike-ins to remove technical bias ...
7
votes
2answers
1k views

Which measure should be used in a PCA or RNA-seq data? TPM or counts?

I'm trying to understand the magnitude of batch effects in my RNA-seq samples, and I was wondering which expression units are more suitable to draw a PCA. I'm thinking of either ...
5
votes
1answer
335 views

Single-cell RNA sequencing (scRNA-seq): filtering cells by transcript counts, how to choose cutoffs?

I am running a notebook with example for the MAGIC algorithm. In the data preprocessing step, a filtering operation is required to filter out cells with a small count of transcripts. My question is ...
4
votes
3answers
355 views

Analysis of differential transcript usage (DTU)

Recent breakthroughs in bioinformatics tools for quantification (e.g. Cufflinks/Kallisto/Salmon etc.) and tools which can identify differential transcript usage (DTU) (e.g. DRIMSeq, Cufflinks etc.) ...
2
votes
1answer
1k views

Cellranger aggr and Seurat merge difference?

I have multiple libraries of 10x Chromium single-cell RNA-seq data, which I'd like to combine. One option is using cellranger aggr which by default does a depth ...
10
votes
2answers
417 views

Can I model technical replicates in DESeq2?

I’d normally use collapseReplicates (or do the collapsing upstream) to handle technical replicates. However, in my current RNA-seq experimental design, samples ...
7
votes
2answers
777 views

Stranded vs. unstranded library preparation protocols in RNAseq

I've been reading this paper lately: Sailfish Enables Alignment-Free Isoform Quantification from RNA-Seq Reads Using Lightweight Algorithms I don't really understand the second paragraph under ...
6
votes
3answers
115 views

How can I compute gene expression for a set of RNA reads?

I'm trying to compute a gene expression profile for an organism. I have gene nucleotide sequences of the mentioned organism stored in a fasta file and a set of paired reads stored in two separate ...
5
votes
1answer
269 views

cell type specific genes - heatmap using rank based approach

I was wondering if some one can help me with pseudo r code for my approach here based on this paper to plot a heatmap Figure 3 Figure description The top 40 enriched genes percelltypeare shown in a ...
5
votes
1answer
70 views

How to compute the chance of failing to detect a gene given the detection limit of a protocol

In Shapiro et al., when discussing about loss of molecules as source of error in single-cell sequencing, it is written that: Another source of error is losses, which can be severe. The detection ...
4
votes
3answers
485 views

Issues performing variant calling with GATK

I am trying to perform variant calling on a BAM file generated through STAR version STAR_2.6.0b for wheat genome using GATK haplotypecaller as follows: ...
4
votes
1answer
839 views

Read counts from BAM file

I have few BAM files which are generated from Ion Torrent Server (ampliseq) aligned to hg19 genome. I want to extract read counts from the bam files and I know that "featureCounts" can be used for ...
4
votes
3answers
228 views

Expression of a gene in different groups

I would like to check the expression of a gene in different groups like Disease vs Normal samples. I want to make a plot out of that to check whether it is significant or not. From this paper lncRNA ...
3
votes
1answer
103 views

How should I address batch effects in my experiment?

Let's say I have an RNA-Seq experiment, where I'm interested in the significantly differentiated genes between pre-treatment and post-treatment conditions. "rep" == biological replicate. ...
3
votes
1answer
235 views

Large dataset normalization for PCA

I need to normalize a large (400Mb) dataset for doing PCA analysis. I want to use scran for doing that: ...
3
votes
2answers
650 views

Error creating indices using STAR

I am trying to index wheat genome using STAR through following command ...
1
vote
2answers
1k views

How to filter ribosomal RNA from scRNA-seq data

I want to filter out ribosomal RNA from scRNA-seq data (downloaded from here). Is there a list of known ribosomal RNA? The only solution I found is SortMeRNA, however it works with raw sequencing ...