Categories
Tag: TPM
Batch and Sample correction for downstream analysis using DESeq2
Hello everyone, I am an absolute beginner on sequencing analysis and DESeq2, so please forgive me for possibly mundane questions. I have tried to look up different methods, but couldn’t find a fitting answer yet. I am currently working with sequencing data derived from an Illumina sequencer. The data is…
A STING Operation in neuroendocrine neoplasms | NANETS2023 | NANETS 2023
1Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; 2University of Minnesota, Minneapolis, MN; 3Caris Life Sciences, Phoenix, AZ; 4The Ohio State University, Columbus, OH; 5Fox Chase Cancer Center, Philadelphia, PA; 6Sylvester Cancer Center, University of Miami, Miami, FL; 7Brown University, Providence, RI Background: Significant advances have been made…
Correlation methods giving very different results (WGCNA)
Hi all, I’ve come back to WGCNA after some years and have run into a bit of a quirky result when looking at my soft power thresholds depending correlation the methods I use. Generally, this topic has been discussed a fair bit – but was looking to see if anyone…
Cibersort input data
Cibersort input data 1 Hello everynone. Could you please guide me. I have an expression matrix that I have filtered and normalized it by edgeR and limma packages. Can I use this for cibersort? Or I have to use PFKM or TPM for cibersort? cibersort • 122 views • link…
Carbohydrates and carbohydrate degradation gene abundance and transcription in Atlantic waters of the Arctic
Seawater samples were collected from surface waters (SRF) and the bottom of the surface mixed layer (BML) in the Eastern Fram Strait region to investigate the distribution of carbohydrates and their utilisation by microbial communities. The ten sites were grouped into three categories based on the underlying seafloor topography (above-slope,…
Chapter 6 GGHH 2023 – notes – Chapter 6. Expression Quantitative Trait Loci (eQTL) Learning Outcomes
Chapter 6. Expression Quantitative Trait Loci (eQTL) Learning Outcomes Define an eQTL Summarise the methodology of RNAseq Understand the reason for expressing RNAseq outcomes as transcripts per million (TPM) Explain why patterns of H3K4me3 and H3K27ac can be used as markers of transcriptionally active genes Incorporate this data into a…
From TPM to raw counts
From TPM to raw counts 0 I am deconvoluting a bulk RNASeq experiment using scRNA to generate a signature of cell types using CIBERSORTX. The program asks you bulk data normalized, so I used TPM. The finction ‘high resolution’ returns normalized expressione (I presume) per cell type. To perform differential…
Solved The following questions are based on the following
The following questions are based on the following paper: Integration of human adipocyte chromosomal interactions with adipose gene expression prioritizes obesity-related genes from GWAS. Nat Commun. 2018; 9:1512. (PMID: 29666371) (i) How is a cis-expression quantitative trait locus (cis-eQTL) defined? (ii) What experimental technique for detecting chromosomal interactions was applied…
Is there a need to batch correct FPKM or TPM values for within sample comparison
Is there a need to batch correct FPKM or TPM values for within sample comparison 0 Hi all, Does anyone have any insight or experience into whether FPKM/TPM expression should be corrected for batch effect? I have come to notice that batch correction is mostly applied to raw counts and…
Using ggplot2 to make barplots of RNASeq data
Using ggplot2 to make barplots of RNASeq data – maintaining sample metadata when pivoting from wide to long format 0 I am currently trying to replicate the following plots of my RNASeq data made by the program Biolayout using ggplot2. This is a network analysis tool which clusters together genes…
How should I run ssgsea analysis ?
How should I run ssgsea analysis ? 1 I have TPM expression data from RNA-seq data analysis. The data comprises of not only protein coding genes but also several other biotypes like miRNA, lncRNA, pseudogene etc making the matrix genes around 60,000. Here, should I filter by data with biotype=”protein…
using RSEM with non Trinity assembly
using RSEM with non Trinity assembly 0 Hi all, I am trying to use RSEM to receive relative abundance estimates of viruses within my metagenomic data, not transcripts. The problem is that I am no longer using Trinity as the assembler because I had much better luck with SPAdes, especially…
DEseq2 input
DEseq2 input 1 Hello Guys, @Michael Love I have a transcriptomics dataset and did rnaseq/nf-core pipeline by salmon-star. my output of the salmon-star folder is as follows: salmon.merged.gene_counts.tsv salmon.merged.gene_counts_length_scaled.tsv salmon.merged.gene_counts_scaled.tsv salmon.merged.gene_lengths.tsv salmon.merged.gene_tpm.tsv salmon.merged.transcript_counts.tsv salmon.merged.transcript_lengths.tsv salmon.merged.transcript_tpm.tsv tx2gene.tsv my question is: which one of these files should be an input for Deseq2…
Multi-mapped reads in Ribo-Seq data, discard or keep?
Multi-mapped reads in Ribo-Seq data, discard or keep? 0 Hi all, I am for the first time doing a TE analysis using Ribo-seq and RNA-seq data, however I have a few question regarding the analysis. I have used STAR to align the reads from both datasets to the Human genome….
Convert FPKM to TPM in R
I’m conducting a meta-analysis over several datasets. I want to combine those datasets and run some machine learning algorithms to predict a target response. Some of those datasets are raw counts, which I can easily convert to TPM with the following code: rpkm <- apply(X = subset(counts_data), MARGIN = 2,…
salmon for gene expression quantification
salmon for gene expression quantification 0 Are the results of salmon reliable for gene expression quantification? Because it just gives gene expression in terms of TPM and Number of Reads by just one command taking input as fastq files. salmon gene-expression • 62 views • link updated 8 minutes ago…
Mitochondrial genes – TPM calculation bulk RNA-Seq
Mitochondrial genes – TPM calculation bulk RNA-Seq 0 Hello all, I was wondering if any of you have encountered a situation for bulk RNA-Seq where, possibly due to low sample quality or presence of dead cells, mitochondrial genes are expressed to a very large degree relative to other genes, thus…
Effect of Bootstrapping/Gibbs Sampling in Salmon Counts
Effect of Bootstrapping/Gibbs Sampling in Salmon Counts 2 Hi Everyone, I am a bit confused about the difference between Gibbs Sampling and Bootstrapping when it comes to Salmon and how these procedures affect downstream analysis. For context, I am trying to do analysis of 49 matched cancer vs. normal RNAseq…
Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens
Developing an epigenetic screening platform in human T cells Staphylococcus aureus Cas9 (SaCas9) has been extensively used for genome editing in vivo as its compact size (3,159 bp) relative to the conventional Streptococcus pyogenes Cas9 (SpCas9) enables packaging into adeno-associated virus26,27,28. However, SaCas9 has not been widely used for targeted gene…
DNA hypomethylation characterizes genes encoding tissue-dominant functional proteins in liver and skeletal muscle
Overview of this study In this study, we measured the DNA methylome from mouse liver and skeletal muscle, integrated the data with the transcriptome and proteome of these mouse tissues22,23, and examined how tissue-dominant protein and gene expression were associated with DNA hypomethylation (Fig. 1). In this study, we measured DNA…
Pearson correlation for RNAseq data
Pearson correlation for RNAseq data – input formats 0 Hi guys, I work with tiny crustaceans and did RNAseq on different species, each with 3 biological replicates (each replicate being a pool of 3 individuals). I want to check if there is a good correlation between my replicates. I trimmed…
Characterization of H3K9me3 and DNA methylation co-marked CpG-rich regions during mouse development | BMC Genomics
CHMs are stable during mouse development To explore the co-localization between H3K9me3 and DNA methylation, we collected public H3K9me3 chromatin immunoprecipitation sequencing (ChIP-seq) and whole-genome bisulfite sequencing (WGBS) data during mouse pre-implantation embryogenesis [11], PGC development [12], spermatogenesis [13, 14], retina development [15], heart and liver development after gastrulation [16,17,18]…
Removing all genome annotations from a list of sequences in Geneious
Removing all genome annotations from a list of sequences in Geneious 0 I have a folder in Geneious which contains over a hundred read mapping files, showing rna-seq reads mapping to genomic scaffolds. Each one of these files is also annotated with multiple gff files, which I will use for…
MICA: a multi-omics method to predict gene regulatory networks in early human embryos
Introduction After the fusion of the oocyte and sperm, the zygote undergoes a series of cell divisions until it forms a blastocyst before implantation into the uterus. A human blastocyst is formed of a fluid-filled cavity and ∼200 cells that comprise three distinct cell types: the trophectoderm (TE), which gives…
Converting STAR Gene-level alignment to TPM expression
Converting STAR Gene-level alignment to TPM expression 0 Hi, I have recently performed gene-level alignment with STAR on 20 samples with the parameter –quantMode GeneCounts and –outSAMtype BAM SortedByCoordinate. I have the output files ReadsPerGene.out.tab and Aligned.sortedByCoord.out.bam. From this, how can I generate reliable TPM values with either the sorted…
What can you expect from Ubuntu 23.10?
• Ubuntu 21.10 focuses on stronger security.• It also includes smoother app discovery processes.• In advance of next year’s bigger update, Ubuntu 23.10 offers a surprising amount of additional refinement. On October 12th, Canonical announced the release of Ubuntu 23.10. It’s not exactly “lines around the Apple store” time, but…
Salmon and SPAdes contigs filtering
Salmon and SPAdes contigs filtering 0 Hello! I have a bunch of reference contigs, obtained with SPAdes, and RNA-seq data analyzed with salmon for control and experimental sample (let it be c_ and 2_). For every gene, there are several contigs, differing by length. Here are some tables Control: ~$…
Combining ComBatseq to remove batch effects and fragment size normalization
Combining ComBatseq to remove batch effects and fragment size normalization 0 In order to use a certain R package, I need fragment size adjusted counts such as TPM or FPKM. However, the raw counts are influenced by batch effects and I want to remove batch effects using ComBatseq. Would it…
Normal vs Tumor – Kaplan Meier Survival Analysis
Normal vs Tumor – Kaplan Meier Survival Analysis 0 To perform survival analysis for normal vs tumor cancer sample what kind of rna seq data is to used? => unstranded => stranded_first => stranded_second => tpm_unstrand => fpkm_unstrand => fpkm_uq_unstrand Which of these shoud be used and is there any…
Simulation of undiagnosed patients with novel genetic conditions
Simulated patient initialization We simulate patients for each of the 2134 diseases in Orphanet20 (orphadata.org, accessed October 29, 2019) that do not correspond to a group of clinically heterogeneous disorders (i.e., Orphanet’s “Category” classification31), have at least one associated phenotype, and have at least one causal gene. For Orphanet diseases…
DESeq2 gene length normalisation
Dear Prof. Love, I was wondering if you could help me with a query about the DESeq2 package in R please. I have sequenced RNA transcripts from six separate species and am comparing gene expression between pairs of species. The issue I am facing is that I cannot work out…
Immunosuppression causes dynamic changes in expression QTLs in psoriatic skin
Mapping eQTLs in patients with psoriasis We obtained longitudinal lesional and non-lesional skin biopsies from participants at baseline, during treatment, and at the time of psoriasis relapse after study medication withdrawal over a course of 22 months. We used genome-wide genotyping and RNA-seq to assay samples. After stringent quality control,…
Bulk RNAseq Standard Data Processing Pipelines
Pipelines and parameters used to process data on the BioBox platform Pipeline for processing public data to sample gene counts SRA-Toolkit is used to fetch the raw files using fasterq-dump -e 3 The files are passed to Kallisto for quantification using kallisto quant -t 3 If the sample is…
TPM from STAR output without re-allign the file using RSEM or Salmon
Hi, I want to get the TPM files from aligned files generate with STAR and reading I found out that the easiest way is using RSEM or Salmon. My code for the alignment is /Users/c/STAR/bin/MacOSX_x86_64/STAR runThreadN 4 –genomeDir /Users/c/Desktop/Human_genome_index –readFilesIn /Users/c/Desktop/test/C1D20_R1_001_paired.fastq /Users/c/Desktop/test/C1D20_R2_001_paired.fastq –quantMode TranscriptomeSAM GeneCounts –outFileNamePrefix C1D20 –outSAMtype BAM SortedByCoordinate…
TPM RNA-seq data for differential expression analysis
TPM RNA-seq data for differential expression analysis 1 Hello. I have a project where I need to identify differences in gene expression between two categories of cancer patients: low and high survival rate. I am retrieving RNA seq data from the Cancer Genome Atlas TCGA, but I can only find…
CollectRnaSeqMetrics (Picard) output to convert FeatureCounts into TPM
CollectRnaSeqMetrics (Picard) output to convert FeatureCounts into TPM 0 Hi, I have bulkRNAseq dates (12 samples, pair end sequenced) and my pipine was : I performed quality control with FastqQC, Trimmed reads with Trimmomatic Aligned reads to the reference genome with STAR Used Samtools to sort and index the BAM…
Calculating TPM from featureCounts output
Calculating TPM from featureCounts output 3 Hi all, Have a simple question but just want to double check I’m not doing something stupid. I have paired-end RNA-seq data for which I have used featureCounts to quantify raw counts. I now want to normalize using the TPM formula. I read this…
Is it possible to convert 3 prime sequencing read counts into TPMs?
Is it possible to convert 3 prime sequencing read counts into TPMs? 1 I have got read counts from 3 prime sequencing and would like to make a rough comparison with other RNAseq dataset for which have got transcripts per million (TPM) values. Is it possible to convert the read…
eQTL mapping in Brown Swiss bulls to identify variants associated with male fertility
Abstract Fertility is an essential component of the livestock industry. In cattle, numerous QTL for male reproductive success fall within regulatory regions. However, the effects of these loci have not been investigated in detail or on a large scale. Here, we assemble a sizeable cohort of mature bulls to detect…
Final Evaluation for Health Recovery in Northeast Syria Phase 3 (HERNES 3) – Syrian Arab Republic
BACKGROUND 1. Context Northeast Syria is a region with a complex and dynamic socio-political landscape. It is situated in the northern part of Syria and bordered by Turkey to the north, Iraq to the east, and the Syrian Desert to the south. Humanitarian challenges in Northeast Syria are exacerbated by…
DNA-bridging by an archaeal histone variant via a unique tetramerisation interface
Chromatin isolation and MNase digestion M. jannaschii DSM 2661 cells were grown in 100 l fermenters in minimal medium containing 0.3 mM K2HPO4, 0.4 mM KH2PO4, 3.6 mM KCl, 0.4 M NaCl, 10 mM NaHCO3, 2.5 mM CaCl2, 38 mM MgCl2, 22 mM NH4Cl, 31 µM Fe(NH4)2(SO4)2, 1 mM C6H9NO6, 1.2 µM MgSO4, 0.4 mM CuSO4, 0.3 µM MnSO4, 36 nM FeSO4, 36 nM CoSO4, 3.5 nM…
SL-scan identifies synthetic lethal interactions in cancer using metabolic networks
Datasets The gene expression data, mutation data, CRISPR, and drug perturbation data sets used in this study were obtained from the Depmap project depmap.org/portal/download/all/. The gene expression data set consists of the log2 transformed transcript per million (TPM) values of 19,221 protein-coding genes from 1406 cell lines across 33 cancer…
kallisto normalized TPM values without bootstraps
Hi everyone! I have quantified the RNA expression in a large number of samples using kallisto, however, I did not include any bootstrapping in my quantifications since I conducted DE analysis using DESeq2, which can’t make use of that information anyways. I wanted to supplement my DE analysis with cell…
Fibroblast Growth Factor Receptor 2 (FGFR2), a New Gene Involved in the Genesis of Autism Spectrum Disorder
The ASD patient described here showed a mutation in the FGFR2 gene, located in the chromosomal band 10q26 (Fig. 1a) and encoding the fibroblast growth factor receptor type 2. It belongs to the family of tyrosine kinase receptors (including FGFR1, FGFR3, and FGFR4) that regulate several biological processes, including bone development,…
Ecophysiology and interactions of a taurine-respiring bacterium in the mouse gut
A taurine-respiring bacterium isolated from the murine gut represents a new genus of the family Desulfovibrionaceae Strain LT0009 was enriched from mouse cecum and colon using an anoxic, non-reducing, modified Desulfovibrio medium with L-lactate and pyruvate as electron donors (and carbon source) and taurine as the sulfite donor for sulfite…
Bioconductor – Linnorm
DOI: 10.18129/B9.bioc.Linnorm This package is for version 3.16 of Bioconductor; for the stable, up-to-date release version, see Linnorm. Linear model and normality based normalization and transformation method (Linnorm) Bioconductor version: 3.16 Linnorm is an algorithm for normalizing and transforming RNA-seq, single cell RNA-seq, ChIP-seq count data or any large…
Bioconductor – zFPKM
DOI: 10.18129/B9.bioc.zFPKM This package is for version 3.16 of Bioconductor; for the stable, up-to-date release version, see zFPKM. A suite of functions to facilitate zFPKM transformations Bioconductor version: 3.16 Perform the zFPKM transform on RNA-seq FPKM data. This algorithm is based on the publication by Hart et al., 2013…
Geo2r rna seq analysis
Geo2r rna seq analysis 0 Hello guys, I am relative new to rna seq data analysis. I would appreciate if anyone could ask my question.. To my understanding after u run rna seq analysis pipeline the information u get is whether the group under investigation ( lets call it disease…
PanCanAtlas EBPlusPlus-corrected RNA-seq TCGA dataset
PanCanAtlas EBPlusPlus-corrected RNA-seq TCGA dataset 4 Hi, I am wondering in which normalisation format (RPKM, FPKM, TPM,… etc) the PanCanAtlas EBPlusPlus-corrected RNA-seq TCGA dataset (the EBPlusPlusAdjustPANCAN_IlluminaHiSeq_RNASeqV2.geneExp.tsv file available here) is in? I know it is batch-corrected, but I don’t know in which normalisation format the original data was in. Thanks…
Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions
Reactor performance revealed temporal changes in propionate degradation rate The four propionate- and acetate-fed reactors used in the study produced biogas with an average methane content of 62–70% (Table S2). The pH was 8.1–8.3, resulting in an ammonia-nitrogen level of 0.7–0.9 g NH3 L-1. This free ammonia level is well above…
prediction dead time within 24 hours with TPM-rna seq data
prediction dead time within 24 hours with TPM-rna seq data 0 Is there a way to know the specific time of death within 24 hours of a patient using TPM rna-seq data in Julia, R, and Python? I’m currently trying to do that analysis with Julia’s CYCLOPS package, but I’m…
Genome-resolved correlation mapping links microbial community structure to metabolic interactions driving methane production from wastewater
Lulu Island waste resource recovery ecosystem The Lulu Island WWTP operated by Metro Vancouver in Richmond, British Columbia, Canada (Longitude: −123.14498° or 123° 8’ 42” W, Latitude: 49.11491° or 49° 6’ 54” N) provides primary and secondary treatment of >30 billion liters of mixed-sourced wastewater from ~200,000 residents each year….
Requesting further clarification on interpreting relative gene expression strength
Requesting further clarification on interpreting relative gene expression strength 0 What is your opinion on the following paragraph in section 6.3 of RNA-seq workflow. (master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html) by the creators of DESeq2 package. Paragraph: “The heatmap becomes more interesting if we do not look at absolute expression strength but rather at the…
Is it valid to do a coverage normalization in addition to applying a spike-in-derived scaling factor?
ChIP-seq visualization: Is it valid to do a coverage normalization in addition to applying a spike-in-derived scaling factor? 0 Let’s say a ChIP-seq sample is scaled in the following way: Calculate the percent spike-in reads in the immunoprecipitate (IP) Calculate the percent spike-in reads in the input Calculate the scaling…
Differential Expression using Isoseq-supplemented reference transcriptome
Differential Expression using Isoseq-supplemented reference transcriptome 1 Hi all, I have a dataset of Illumina short read RNA-Seq data from (n = 6 per group) three different mouse genotypes, and paired PacBio Isoseq data from a subset of these (n = 2 per group). I have processed the IsoSeq data…
Solanum americanum genome-assisted discovery of immune receptors that detect potato late blight pathogen effectors
Genome assembly and gene model prediction of S. americanum S. americanum is a globally distributed Solanaceae species that is resistant to many pathogens, including P. infestans and Ralstonia solanacearum25,29,33. Four S. americanum accessions SP1102, SP2271, SP2273 and SP2275 were selected for sequencing based on their variation in resistance to late…
Limma couldn’t find the differential gene
Limma couldn’t find the differential gene 1 Hi, I am using limma for differential gene analysis of RNA seq results. I have encountered the following issues: I cannot find any genes with significant padj values (less than 0.05). Although some genes have extremely high logFC values. (I use TPM values…
Creating tx2gene table from sheep transcripts IDs to gene IDs using NCBI annotation file
Creating tx2gene table from sheep transcripts IDs to gene IDs using NCBI annotation file 0 Hello, I am trying to create a tx2gene table using NCBI annotation file (GCF_016772045.1) ramb_gtf <- import(“genomic.gtf”) txdb <- makeTxDbFromGRanges(ramb_gtf) k <- keys(txdb, keytype = “GENEID”) df <- select(txdb, keys = k, columns = “TXNAME”,…
Genome-wide analysis and characterization of the LRR-RLK gene family provides insights into anthracnose resistance in common bean
Identification of PvLRR-RLK genes From the kinome of P. vulgaris30, 1203 PKs were identified. Of these, only the proteins endowed with the transmembrane kinase and LRR domains were retained (Supplementary Table S1). All PvLRR-RLKs obtained were analyzed for redundancy following the criterion of maintaining the largest variants in the case…
Remove batch effects on the train set to avoid information leakage
Remove batch effects on the train set to avoid information leakage 0 I aim to apply Limma’s removeBatchEffect function on my data, but only after splitting it into train and test sets. I’m aware that applying batch correction before this partition can introduce information leakage, so I want to avoid…
Performing differential expression analysis after applying transformations on my data
Performing differential expression analysis after applying transformations on my data 1 I possess RNA-seq data that’s TPM normalized, sourced from different origins. I merged these datasets and then applied log2 transformation followed by batch effect correction. These steps ensured that all samples approximated a similar range, making them crucial for…
Jointly analyzing DEGs – my RNA-seq and GTEx
Jointly analyzing DEGs – my RNA-seq and GTEx – How to do it properly? 0 I have some standard polyA RNA-seq data from whole human tissue that I generated. I want to compare the relative abundance of genes in my tissue dataset with all the other tissues of the body….
About rnaseq_salmon
RNAseq Salmon (rnaseq_salmon) is a DNAnexus Workflow that combines the two DNAnexus applets Salmon Scatter-Process-Gather Workflow and quant_sf2express_table. Salmon Scatter-Process-Gather Workflow (salmon_spg_wf) is a DNAnexus applet that process a batch of pair-end FASTQ read files and runs Salmon to produce expression count files. quant_sf2express_table is a DNAnexus applet that generates expression table files suitable for RNA-seq Expression…
log2Fold Change as input for k-means analysis
log2Fold Change as input for k-means analysis 0 Hi, I am analyzing a RNA-seq dataset that include different treatments and time points. I would like to perform a k-means analysis to cluster my genes. I have already performed a differential expression analysis with DESeq2 and I selected the significantly up-regulated…
Make heatmap for RNA-seq with non replicate
Make heatmap for RNA-seq with non replicate 0 Hi all, degs = rownames(subset(DEG, PValue < 0.05 & abs(logFC > 9))) rownames(counts) = DEG[rownames(counts), ‘symbol’] counts_degs = counts[degs,] pheatmap(counts_degs, clustering_method = ‘ward.D’, scale=”row”) Could I use TPM matrix instead of raw count matrix to make heat map using the code above?…
Calculating FPKM and TPM by hand from htseq-count output?
Calculating FPKM and TPM by hand from htseq-count output? 0 Hello! I am counting reads with htseq-count, and wasted some hours trying to find an extant software that would calculate FPKM and/or TPM from that output, so I wrote a script myself. There is just one question mark – should…
How to interpret heatmap using plotheatmap from deeptools?
How to interpret heatmap using plotheatmap from deeptools? 1 The heatmap indicates by color the amount of signal (whatever this is, RPKM, TPM, normalized counts…) in a windows of +/- 3000bp around the TSS which is the center. The more blue the more signal, the more red the less signal….
How to best represent three biological replicates of single tissue sample?
How to best represent three biological replicates of single tissue sample? 1 Hello All, Curious as to how to best represent three biological replicates of a single tissue sample of RNASeq data? Assuming that these each replicates are normalized (RPKM, TPM), is there any other statistical analysis that needs to…
error with Tximport when txOut = TRUE
Hello everyone, Hello Everyone, I am having issue with when trying to aggregate transcript abundances to the gene level (when txOut=FALSE) but it works fine with txOut=TRUE. Here are the steps I followed: Produced bam file using Gencode transcript fasta file. Further sorted and index them. Used Nanocount to produce…
Revisit where to find CCLE RNAseq in FPKM or RPKM using RSEM values to perform normalization- as was never answered usefully
Revisit where to find CCLE RNAseq in FPKM or RPKM using RSEM values to perform normalization- as was never answered usefully 0 I would like to find the CCLE RNA expression file that has either effective gene sizes or FPKM /RPKM (where estimated RSEM values have been used) to do…
Is RSEM required for RNA-seq data analysis using STAR and edgeR?
Is RSEM required for RNA-seq data analysis using STAR and edgeR? 1 I have fastq files (n=40) obtained by paired-end unstranded RNA-seq. I would like to analyze these files using STAR for mapping and perform the differential expression analysis between the two groups by edgeR. I plan to apply TPM…
Need a tutor for DESeq analysis
Need a tutor for DESeq analysis 0 Through various sources, I have tried to do DESeq analysis and functional profiling. As this is my first time doing this, I want someone to go through my R code and give me some suggestions as I am stuck at certain places. tutor…
Global within-species phylogenetics of sewage microbes suggest that local adaptation shapes geographical bacterial clustering
Predominant bacteria in sewage do likely not originate from the human gut To identify bacterial genomes from sewage across the world, we used a combination of two different metagenomics genome binners (VAMB24 and MetaBAT225). From 757 samples across 101 different countries (Fig. 1a and Supplementary Fig. 1), we were able to create…
Antisense therapy restores fragile X protein production in human cells
(A) Volcano plot of log2FC of RNA levels (FXS vs TD). Statistically significant changes (P value <0.0002) are shown as blue dots (down-regulated) and red dots (up-regulated). Gray dots refer to unchanged RNAs. (See also Dataset S2). (B) Histograms for TPM values for RNAs that are up or downregulated in…
How reproducible is transcript quantification through salmon?
How reproducible is transcript quantification through salmon? 0 Hello! I am conducting differential expression analysis on a subset of plant transcripts. I have decided to go with Salmon+tximport+DESeq2. I am using the pseudoalignment mode of salmon on fastp-trimmed fastq files. Salmon index is run with ‘-k=31’ and quant is run…
the problem with rpkm (and tpm)
Can you please explain the main core problem with RPKM normalization (as a measure of relative abundance), using a simple example, and why TPM solves this? Different explanations for why the RPKM unit is bad are: (a) it uses length normalization, (b) it normalizes to total library size, (c) because…
Download | RatGTEx
See the About page for processing and data format specifications. Gene info Gene expression Median TPM per gene per tissue Used for heatmap visualizations log2(count+1) Used to compute allelic fold change Adipose | BLA | Brain | Eye | IL | LHb | Liver | NAcc | NAcc2 | OFC…
No expression found with Salmon and Kallisto
Hi everyone ! I have a small nucleotid sequence (24nt) of which I know the location in the human genome and I have a rna-seq transcriptome. I already know that my sequence is expressed in this transcriptome because I found 5 hits for it with a grep. However, I used…
CIBERSORTx/fractions docker outputs only zeros if –absolute TRUE
CIBERSORTx/fractions docker outputs only zeros if –absolute TRUE 0 Hey everyone, I am using the docker container for CIBERSORTx/fractions. I would like to run it in absolute mode (since from this I can also calculate the relative numbers). This guy also had some trouble using it, but it seems different…
TPM normalization but library size isn’t equal to 1 million
TPM normalization but library size isn’t equal to 1 million 0 I have downloaded a bulk RNA-seq data frame for this article. All data was downloaded from here. In supplementary_table_4, the first sheet is READ ME, and it says that sheet 8 is “Raw RNA-seq log2(TPM+1) values for all 261…
Wnt signaling preserves progenitor cell multipotency during adipose tissue development
Acute transcriptional remodeling upon adipogenic stimulation We used a previously described method to generate multipotent progenitor cells from human adult adipose tissues24. All procedures were conducted in accordance with the UMass Chan Institutional Review Board ID 14734_13. Briefly, small fragments of subcutaneous adipose tissue destined to be discarded from individuals…
Using IMmuno-PREdictive Score (IMPRES)
Using IMmuno-PREdictive Score (IMPRES) 0 I have multiple bulk RNA-seq datasets, all of which have been normalized to TPM. However, one or two of these datasets have undergone different normalization techniques. To standardize the scale across all datasets, I performed upper quartile normalization (UQN) followed by a log2 transformation. I…
How do I run DE for TPM values (not CPM)?
How do I run DE for TPM values (not CPM)? 1 Can someone explain to me how to run DE using TPM instead of CPM, please? All the DE guides I’m seeing only use CPM values, but I need to work with TPM values. Providing either a guide of code…
Filtering genes after TPM normalization
Filtering genes after TPM normalization 0 Filtering genes is one of the steps that I do in all my analysis. I usually filter the counts RNA-seq data according to a specific threshold. In my current work, I have no access to the counts data, I only get the normalized data….
Using Tumor Immune Dysfunction and Exclusion (TIDE)
Using Tumor Immune Dysfunction and Exclusion (TIDE) 0 I have multiple datasets with bulk RNA-seq and the metadata for response / no response to immune checkpoint blockade therapy. I want to use Tumor Immune Dysfunction and Exclusion, TIDE, in order to use the transcriptomic data, to predict response. In order…
How do I calculate differential expression for RNA-seq values with the “limma” package and the “ebayes” function?
How do I calculate differential expression for RNA-seq values with the “limma” package and the “ebayes” function? 0 So for context, I have a set of TPM values (which I converted to log2(TPM+1) for multiple genes for different samples, and I need to calculate the differential expression for RNA-seq values….
Adjusting for batch effect and covariates with ComBat
Dear All, my question is related to this post: Error in while (change > conv) { : missing value where TRUE/FALSE needed I have a heterogeneous RNAseq dataset in TPMs from 66 samples and two sequencing batches (64 from one batch, 2 from the second batch). This dataset contains many…
Figures and data in Endoparasitoid lifestyle promotes endogenization and domestication of dsDNA viruses
The panel (A) refers to the four known cases (Venturia canescens, Fopius arisanus, Cotesia congregata, and Microplitis demolitor) involving Nudivirus donors while the panel (B) refers to the known case involving LbFV donors in three Leptopilina species. Complete parasitoid wasp genomes information was available for Microplitis demolitor, Venturia canescens, Fopius…
How to calculate TPM from featureCounts output
How to calculate TPM from featureCounts output 0 I would like to find the TPM counts for the GSE102073 study. When i downloaded the raw data from GEO, the raw data are featureCounts output. First part of the file: # Program:featureCounts v1.4.3-p1; Command:”/data/NYGC/Software/Subread/subread-1.4.3-p1-Linux-x86_64/bin/featureCounts” “-s” “2” “-a” “/data/NYGC/Resources/ENCODE/Gencode/gencode.v18.annotation.gtf” “-o” “/data/analysis/LevineD/Project_LEV_01204_RNA_2014-01-30/Sample_JB4853/featureCounts/Sample_JB4853_counts.txt” “/data/analysis/LevineD/Project_LEV_01204_RNA_2014-01-30/Sample_JB4853/STAR_alignment/Sample_JB4853_Aligned.out.WithReadGroup.sorted.bam”…
LinkedOmics :: Data Download
RNAseq (HiSeq, Gene level, Tumor) Download RNAseq data RSEM upper-quartile normalized (Illumina HiSeq platform, Gene-level) gene Expression (RSEM-UQ, Log2(Val+1)) 140 28057 cct RNAseq (HiSeq, Gene level, Normal) Download RNAseq data RSEM upper-quartile normalized (Illumina HiSeq platform, Gene-level) gene Expression (RSEM-UQ, Log2(Val+1)) 21 28057 cct RNAseq (HiSeq, Gene level, Duct) Download…
many over and under-expressed features in modules of a signed network
WGCNA: many over and under-expressed features in modules of a signed network 1 In a WGCNA analysis of transcriptome and proteome of a white blood cell in development (in 6 stages), I find in most modules (especially the large ones) over, as well as underexpressed features, but I am using…
What type of normalization did they use in this article?
What type of normalization did they use in this article? 1 I’ve read this amazing article, yet I’m struggling to understand how did they normalize the bulk RNA-seq data. I’ve downloaded the data from the supplementary information, and the values of all the genes are around 30, so this can’t…
Graphing Average Expression of Group of Genes
Graphing Average Expression of Group of Genes 0 I’m trying to plot the expression of a group of genes over time. My starting dataframe is in TPM like below. V1 MEF DAY3 DAY6 DAY9 DAY12 IPSC Arhgef12 48.9061752 76.001558 64.294236 61.0208545 66.4678191 25.11639309 Arnt2 8.6570850 11.341789 22.613930 35.2099605 36.0336247 1.30568627…
What is the best way to clean bulk RNA-seq data?
What is the best way to clean bulk RNA-seq data? 1 As far as I know, there isn’t a universally agreed-upon threshold or an approach to clean the data. I want to remove the genes that don’t contribute, or in other words, the noise genes, BEFORE I normalize the data,…
ASX Health Stocks: Tissue Repair, Radiopharm jump double digits after US FDA meetings
Two ASX health stocks had double-digit share price jumps on Monday morning, after positive US Food and Drug Administration moves. Meanwhile two other companies have also delivered good news. For the latest health news, sign up here for free Stockhead daily newsletters Tissue Repair to progress to Phase 3 Wound…
Does the RNAseq data normal if the TPM value 3rd Qutile expression is near 10, but the Max expression are near 20,000
Does the RNAseq data normal if the TPM value 3rd Qutile expression is near 10, but the Max expression are near 20,000 1 Dear all, May I have your guidance that the gene expression TPM as below is normal? The most highly expressed seems all correlated with Ribonucle, if filter…
Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri | Parasites & Vectors
Mapping of bulk RNA-seq data and differential gene expression (DGE) Using the splice-aware aligner STAR, we mapped the RNA-seq reads to the H. bakeri genome assembly obtained from WormBase ParaSite (PRJEB15396). Among all the datasets, 93.26–95.62% of the reads uniquely mapped to the reference genome (Table 1), reflecting the high…
Comparing gene expression with copy number variation in TCGA
Hello, I want to compare (with a PCA) gene expression against copy number variation at gene level in a TCGA project.When I retrieve the gene expression every value is mapped by sample and gene. But for the copy number variation, I get only chromosomal locations.To do the PCA, I want…
Is the gene-specific PCR efficiency a serious concern for intrasample comparisons in RNA-Seq?
Is the gene-specific PCR efficiency a serious concern for intrasample comparisons in RNA-Seq? 1 Or “Everything You Always Wanted to Know About RNA-Seq (But Were Afraid to Ask) Part 2” In RNA-Seq, it is common practice to compare the abundance of transcripts within the same sample after some form of…
Normalizing RNA read counts for each gene?
Normalizing RNA read counts for each gene? 2 While reading a paper, I encountered the line “Heatmap of normalized read counts for chlorophyll-protein complexes (LHCs) normalized by row to reflect relative expression of each gene (see scale bar)” and was wondering if someone would be able to clarify how the…