Tag: RPKM

Merge multiple text files to create a combined dataframe and rename columns in R – General

Hi, I have multiple .txt files (each file contains 4 columns; an identifier Gene column, a raw_counts and other columns). I would like to merge those files into a combined dataframe using the common gene column. I was able to import multiple .txt files together, merge based on identifier column,…

Continue Reading Merge multiple text files to create a combined dataframe and rename columns in R – General

Metagenomics – RPKM calculation

Normalization for comparing gene coverage values. RPKM corrects differences in both: sample sequencing depth and gene length. RPKM – Reads per kilo base per million mapped reads Formula RPKM = numReads / ( geneLength/1000 * totalNumReads/1,000,000 ) numReads – number of reads mapped to a gene sequence geneLength – length…

Continue Reading Metagenomics – RPKM calculation

a strange pattern of repetitive summits

Problem with the output of Deeptools PlotProfile: a strange pattern of repetitive summits 0 Hi! I am trying to plot DNA binding profiles of my ChIP-seq bw files using Deeptools plotProfile. I generated the matrix using the computeMatrix reference-point. I used some publicly available bed files as my regions of…

Continue Reading a strange pattern of repetitive summits

RPKM threshold estimation – SEQanswers

Dear All, I have a doubt in the calculation of False postitive rate while checking for FPKM threshold in a RNAseq experiment. Following the method previously published (www.ploscompbiol.org/article/…l.pcbi.1000598). I am not getting desired results. I followed the method as mentioned the publication Reads were mapped to Ensembl genes (blue) and…

Continue Reading RPKM threshold estimation – SEQanswers

RPKM, how to normalize for library size

RPKM, how to normalize for library size 1 Hi everybody! I’m working on several metagenomes (so not trascriptomes), and I mapped high quality reads to a database made of nucleotide sequences through bowtie2. I want to convert raw mapping counts of each gene in RPKM. I know this method might…

Continue Reading RPKM, how to normalize for library size

Transcriptional profiling of macrophages reveals distinct parasite stage-driven signatures during early infection by Leishmania donovani

Infection with L. donovani amastigotes or promastigotes promotes early changes in the mRNA pool of the host cell To compare the early effects of the two life stages L. donovani in the mature mRNA pool of the host cell, total cytosolic mRNA extracts from bone marrow-derived macrophage (BMDM) cultures infected…

Continue Reading Transcriptional profiling of macrophages reveals distinct parasite stage-driven signatures during early infection by Leishmania donovani

Can I convert HTSeq count into RPKM or TPM value or standard unit of RNA-Seq

Can I convert HTSeq count into RPKM or TPM value or standard unit of RNA-Seq 0 Now, I’m comparing RNA expressions that have RNA-Seq and HTSeq count How can I interpret it together with different unit or Can I convert HTSeq count equivalent RNA-Seq? or if you have other suggestions,…

Continue Reading Can I convert HTSeq count into RPKM or TPM value or standard unit of RNA-Seq

use tcgabiolinks package to download TCGA data

TCGA Data download in terms of ease of use ,RTCGA The bag should be better , And because it’s already downloaded data , The use is relatively stable . But also because of the downloaded data , There is no guarantee that the data is new .TCGAbiolinks The package is…

Continue Reading use tcgabiolinks package to download TCGA data

How to do functional analysis on differentially expressed gene list from RNA-seq data?

How to do functional analysis on differentially expressed gene list from RNA-seq data? 0 @06f08eeb Last seen 1 day ago Canada Hi all, I am a complete beginner in terms of bioinformatics analysis and I am hoping to complete some functional analysis on some differentially expressed gene lists of some…

Continue Reading How to do functional analysis on differentially expressed gene list from RNA-seq data?

DESeq2 input from GDAC firehose

Hi guys, I hope you are fine. I’m not good in English so if you couldn’t understand my question, please feel free to reply. I’m a beginner of bioinformatics. I want to practice differential expressed gene (DEG) analysis in R. The RNA seq data I used was downloaded from broad…

Continue Reading DESeq2 input from GDAC firehose

How to perform deconvolution with RPKM values

Hi Xuran, I tried to apply MuSiC to RNAseq bulk data with RPKM as the input. According to your paper(Discussion), “MuSiC can utilize RPKM if estimates of cell type-specific total RNA abundance can be provided.” I am wondering how I can incorporate cell-type-specific total RNA abundance into your function? Or…

Continue Reading How to perform deconvolution with RPKM values

r – RNA-Seq Data Heatmap: Is it necessary to do a log2 transformation of RPKM values before doing the Z-score standardisation?

I am making a heatmap using RNA-Seq data in R. The heatmap shows gene expression values (RPKM) in different brain regions. I have the following code: library(tidyverse) library(pheatmap) library(matrixStats) read_csv(“prenatal_heatmap_data.csv”) -> all_data all_data %>% column_to_rownames(“Brain Region”) -> heatmap_data heatmap_data %>% pheatmap() Which generates the following heatmap: I want to do…

Continue Reading r – RNA-Seq Data Heatmap: Is it necessary to do a log2 transformation of RPKM values before doing the Z-score standardisation?

New bioinformatics method to analyze viral sgRNA

Single guide ribonucleic acid (sgRNA) molecules are produced by discontinuous transcription, in which viral RNA-dependant RNA polymerase pauses early negative-sense RNA synthesis and then jumps to the other end of the genome. The specifics of this process are still not fully understood. Since sgRNAs can play an important role in…

Continue Reading New bioinformatics method to analyze viral sgRNA

New bioinformatics method for viral sgRNA analysis

Single guide ribonucleic acid (sgRNA) molecules are produced by discontinuous transcription, in which viral RNA-dependant RNA polymerase pauses early negative-sense RNA synthesis and then jumps to the other end of the genome. The specifics of this process are still not fully understood. Since sgRNAs can play an important role in…

Continue Reading New bioinformatics method for viral sgRNA analysis

Comparing RPKM (calculated from raw counts rnaseq file) between a few genes across 2 experimental conditions with a t-test : bioinformatics

Hello everyone! Very new to analyzing RNAseq data and trying to learn the process behind it. This is completely not my area, but I would like to learn and was hoping someone could give me an explanation to my question. Thank you! I’d like to know if a few genes…

Continue Reading Comparing RPKM (calculated from raw counts rnaseq file) between a few genes across 2 experimental conditions with a t-test : bioinformatics

How to define the gene length for RPKM calculation

How to define the gene length for RPKM calculation 4 Hi guys, I would like to calculate the RPKM of my RNA seq experiment. To do this, as from the formula, I need to know the gene length. My starting point are the row reads (single end) counts resulting from:…

Continue Reading How to define the gene length for RPKM calculation

Frontiers | Metagenomic Analysis Reveals New Microbiota Related to Fiber Digestion in Pigs

Introduction Corn and soybean meal are the main components of high energy and high protein diets for pigs and are also the main raw materials of food products for human consumption, fermentation, and bioenergy industry (Sevillano et al., 2018). As the arable land of food crops was limited whereas the…

Continue Reading Frontiers | Metagenomic Analysis Reveals New Microbiota Related to Fiber Digestion in Pigs

CD19 expression is maintained in DLBCL patients after treatment with tafasitamab plus lenalidomide in the L-MIND study

Introduction CD19 is an important target for novel anti-lymphoma treatments as it is broadly and homogenously expressed across many B-cell malignancies [1,2]. Approximately 30–50% of patients with diffuse large B-cell lymphoma (DLBCL) who do not respond to first-line therapy with R-CHOP have a poor prognosis and need effective treatment options,…

Continue Reading CD19 expression is maintained in DLBCL patients after treatment with tafasitamab plus lenalidomide in the L-MIND study

Using TCGA RSEM data to calculate isoform expression

Using TCGA RSEM data to calculate isoform expression 0 Hello everyone, I have download the TCGA RNAseq RSEM data about isoform expression. I would like to check which one of the isoforms of my gene is the one expressed the most. Can I directly use the RSEM values to conclude…

Continue Reading Using TCGA RSEM data to calculate isoform expression

Is it conventional to use a threshold RPKM value to determine if a gene of interest is expressed in a sample? : bioinformatics

Hello, I am interested in finding out at what stages of development a gene of interest is expressed in the human brain. I am using the Developmental Transcriptome tool from the Allen Brain Atlas to find this information. The heatmap showing the gene expression levels across the different brain samples…

Continue Reading Is it conventional to use a threshold RPKM value to determine if a gene of interest is expressed in a sample? : bioinformatics

Using dgelist function in EdgeR to calculate RPKM

Using dgelist function in EdgeR to calculate RPKM 0 Hi, I’m new to RNAseq and I want to calculate rpkm values from my raw counts but I am unsure if my understanding of how to do so is correct. From what I understand, I should do the following: df <-…

Continue Reading Using dgelist function in EdgeR to calculate RPKM

Differential enrichment of H3K9me3 at annotated satellite DNA repeats in human cell lines and during fetal development in mouse

The removal of problematic regions The removal of problematic genomic regions is considered essential for the accurate analysis of data obtained by chromatin immunoprecipitation followed by genome sequencing (ChIP-Seq) [27, 35]. Repetitive regions including satellite DNA arrays comprise a majority of such problematic regions, mainly because they reside in the…

Continue Reading Differential enrichment of H3K9me3 at annotated satellite DNA repeats in human cell lines and during fetal development in mouse

What is bigwig file?

Asked by: Vada Ratke Score: 4.7/5 (25 votes) BigWig is a file format for display of dense, continuous data in a genome browser track, created by conversion from Wiggle (WIG) format. BigWig format is described at the UCSC Genome Bioinformatics web site, and the Broad Institute file format guide provides…

Continue Reading What is bigwig file?

Analyzing gene expression in different RNAseq datasets

Analyzing gene expression in different RNAseq datasets 0 Hello! I really need some assistance here, I came up with an analysis of my own that makes sense to me but I really new in this (started studying bioinformatics on my own with the pandemics) and I’m not sure if I…

Continue Reading Analyzing gene expression in different RNAseq datasets

Up-to-date RNA-Seq Analysis Training/Courses/Papers (Dec 2017)

Hi all, I am a PhD student with biology background. I recently inherit a RNA Sequencing project from another PhD student in my lab. We already have paired-ended RNA-Seq data generated from Illumina HiSeq but haven’t started analysis yet. I have basic Linux command line training but have no idea about how…

Continue Reading Up-to-date RNA-Seq Analysis Training/Courses/Papers (Dec 2017)

Convert FPKM and RPKM to TPM values

Convert FPKM and RPKM to TPM values 0 Hi all, I’m trying to collect the datasets from public datasets from GEO. I found some data in fpkm and some in rpkm,tpm. My goal is to feed these data into a machine learning model, but my question is “ALL THESE DATA…

Continue Reading Convert FPKM and RPKM to TPM values

ATAC-seq sample normalization

What you describe seems to be a difference in signal-to-noise ratio which is not uncommon. This is where more elaborate normalization methods such as TMM from edgeR or RLE from DESeq2 come into play. See the following links on why these methods are superior. The videos talk about RNA-seq but…

Continue Reading ATAC-seq sample normalization

Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols

Forum:Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols 2 Published in the RNA Journal in 2020 – this paper argues that if the original RNA amount in the different samples is different, TPM should not be used to find differentially expressed genes. www.ncbi.nlm.nih.gov/pmc/articles/PMC7373998/ Seems like…

Continue Reading Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols

Calculating fpkm from raw counts

Calculating fpkm from raw counts 2 Hello, I have a file consisting of raw counts. For my analysis, I want to normalize the counts by fpkm. The question is, how can I calculate fpkm from raw counts? Also, upon a little reading I found that DESeq2 could do i, but…

Continue Reading Calculating fpkm from raw counts

Statistics on RNAseq data

Statistics on RNAseq data 2 Hi I would like to know whether you can do statistical tests (e.g. ANOVAS etc.) on the TPM/RPKM counts of RNAseq data? Thanks on Statistics data RNAseq • 58 views This is not recommended due to a few underlying problems with RNA-seq data that include…

Continue Reading Statistics on RNAseq data

TMM followed by inverse normal transform

TMM followed by inverse normal transform 0 Hey all, I am following a protocol from a paper that uses the following pre-processing procedure: a. Read counts were normalized between samples using TMM (Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome…

Continue Reading TMM followed by inverse normal transform

RPKM on TSS using DiffBind

RPKM on TSS using DiffBind 1 Hi everyone, I want to extrapolate RPKM value from Diffbind. Investigated regions are TSS (± 2.5kb) but around 3500 TSS are “lost” because merged. I have read that in the new version of DiffBind is possible to use dbObj_region$config$mergeOverlap with negative value to avoid…

Continue Reading RPKM on TSS using DiffBind

RPKM and differential expression analysis

RPKM and differential expression analysis 1 Hello all, I am trying to analyse differential expression in a dataset for which I only have RPKM values available to me. I usually use the R/BioC environment for RNA-seq analysis, and have read in various BioC documentation that using RPKM values in packages…

Continue Reading RPKM and differential expression analysis

pre-proccessing of RNAseq data for WGCNA

pre-proccessing of RNAseq data for WGCNA 0 Hi everyone, i wanted to create an expression matrix for WGCNA input. however, i has been said that use RPKM/FPKM data instead of CPM, how can i change my TCGA data to RPKM/FPKM in GDCquery and how to filter expression set of genes…

Continue Reading pre-proccessing of RNAseq data for WGCNA

DESeq2 with a small number of genes

DESeq2 with a small number of genes 1 Dear all, I am writing a program in order to study the coverage of only one sequence. To sum up the pipeline: Detect ORFs in the input sequence Align all reads on the sequence (bowtie), reads come from RNA-seq Count the number…

Continue Reading DESeq2 with a small number of genes