Tag: Spike-Ins

Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases

Reagents miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany, # 217204); TaqMan® Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Bedford, MA, USA, #A28007); TaqMan® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA, # 4444556); TaqMan® Advanced miRNA Assays Single-tube assays (Applied Biosystems, Bedford, MA, USA, # A25576: 478293_mir, Spike-In cel-miR-39-3p;…

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rna seq – How to incorporate negative controls in DESeq2

We are doing a comparison between two outcomes (positive and negative). We could not have any positive controls as we do not have any “control” data to set as baseline, either from literature or otherwise. No spike-ins have been used. We have some samples which had no resolved outcome, hence…

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Rational probe design for efficient rRNA depletion and improved metatranscriptomic analysis of human microbiomes

doi: 10.1186/s12866-023-03037-y. Asako Tan #  1 , Senthil Murugapiran #  2 , Alaya Mikalauskas #  3 , Jeff Koble  4 , Drew Kennedy  4 , Fred Hyde  1 , Victor Ruotti  1 , Emily Law  2 , Jordan Jensen  2 , Gary P Schroth  4 , Jean M Macklaim  3 , Scott Kuersten  5 , Brice LeFrançois  6 , Daryl M Gohl  7   8  …

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Webinar: Metagenome quantitative SIP at JGI

 The DOE Joint Genome Institute offers metagenome quantitative stable isotope probing (Metagenome – qSIP) capabilities to investigate metabolic activity of metagenome-assembled genomes (MAGs). The JGI Metagenome – qSIP workflow begins with users carrying out isotope enrichment culturing and DNA extractions which are sent to the JGI for sample processing….

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RMA with Affymetrix Expression Console vs Oligo package

RMA with Affymetrix Expression Console vs Oligo package 1 I have 192 .CEL files (chip: Affymetrix Human Clariom S HT ). If I apply the RMA using Affymetrix Expression Console software (Analysis: Gene Level – SST-RMA) or with the rma() function from the Oligo package in R Bioconductor, I get…

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Spike-in normalization in ATAC-Seq with DiffBind

Spike-in normalization in ATAC-Seq with DiffBind 0 Dear all, we are planning to do an ATAC-Seq experiment in human cell lines and we must correct for batches from different time-points. Therefore, we want to use spike-in normalization as described in the DiffBind vignette. For reasons of practicability, we would prefer…

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Metagenomic profiling pipelines improve taxonomic classification for 16S amplicon sequencing data

Publicly available mock community sequencing datasets 136 mock community sequencing samples were collected in total from four publicly available sequencing datasets and analyzed in our evaluation. 69 samples are from Lluch et al.33; 33 samples are from Kozich et al.34; 29 samples are from Fouhy et al.35; and 5 samples…

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Absolute Quantification of Microbiota in Shotgun Sequencing Using Host Cells or Spike-Ins

Abstract Background: An ongoing challenge for DNA sequencing of samples containing microorganisms is the ability to meaningfully compare different samples and to connect the results back to clinically relevant disease states. The reads of DNA sequence from each sample do not, in and of themselves, give sufficient information to calculate…

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Problems using ERCC spike-ins for normalization in DESeq2

Hello everyone, I’m analyzing some RNA-seq datasets for differential expression. We did not run the experiments ourselves, but the database where I got them from indicates that they were done adding additional spike-ins (ERCC92) for normalization. Originally, I did not use these spike-ins, and just went along with the default…

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Counting dots or counting reads? Complementary approaches to estimate virus-to-microbe ratios

“How many viruses are there in the environment, compared to cells ?”. This deceptively simple question remains challenging to address today, yet because it relates to fundamental processes and characteristics of viral communities, even the imperfect approximations available have been critical for the field of viral ecology. Most notably, early…

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Using metagenomics to identify viruses in plasma samples

Overview of the study design. We conducted RT-qPCR on 670 plasma samples, followed by metagenomic sequencing of 593 of the samples, received from (i) individuals suspected to have Lassa Fever (LF; caused by Lassa virus, LASV), collected from teaching hospitals with clinical expertise in viral hemorrhagic fevers; (ii) suspected infectious…

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Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA

doi: 10.1186/s12864-023-09541-9. Affiliations Expand Affiliations 1 State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China. shaozhenyu2017@sibcb.ac.cn. 2 Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical…

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nf-core/marsseq

Introduction nf-core/marsseq is a bioinformatics single-cell preprocessing pipeline for MARS-seq v2.0 experiments. MARS-seq is a plate-based technique that can be combined with FACS in order to study rare populations of cells. On top of the pre-existing pipeline, we have developed an RNA velocity workflow that can be used to study…

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Foreign RNA spike-ins enable accurate allele-specific expression analysis at scale

Motivation: Analysis of allele-specific expression is strongly affected by the technical noise present in RNA-seq experiments. Previously, we showed that technical replicates can be used for precise estimates of this noise, and we provided a tool for correction of technical noise in allele-specific expression analysis. This approach is very accurate…

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Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins – PubMed DNA:RNA ImmunoPrecipitation and high-throughput sequencing.

doi: 10.1007/978-1-0716-2477-7_26. Memberships Expand Connections 1 Department about Chemical and Systems Biology, Stanford University, Stand-ford, CANCER, USA. [email protected] 2 Department of Chemical and Systems Nature, Stanford University, Stanford, CA, USA. [email protected] Freely PMC article Item in Clipboard Magdalena P Crossley et aluminium. Methods Mol Bil. 2022. Free PMC books Show details…

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Streamlined quantitative analysis of histone modification abundance at nucleosome-scale resolution with siQ-ChIP version 2.0

In this section, we derive a simplified expression for the proportionality constant \(\alpha\) that enables quantitative ChIP-seq and we introduce some consequences for track building. This new expression is more intuitive to understand, easier to evaluate, and more accurate to sequencing outcomes than the previous expression. While values derived from…

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ERCC normalization within DESeq2

ERCC normalization within DESeq2 0 Hi, I would like to make sure I understand how to do it, if I have a data set with ERCC spike-ins in bulk-RNASeq After integrating the ERCC sequences into the genome (both fastA and gtf) in question, I can just map the fastq files…

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Metagenomic Methods for Addressing NASA’s Planetary Protection Policy Requirements on Future Missions: A Workshop Report

1. Introduction Since the beginning of extraterrestrial exploration by NASA, planetary protection (PP) has been an important effort to prevent biological forward contamination of non-Earth environments. The Committee on Space Research (COSPAR) has formulated a Planetary Protection Policy with associated implementation requirements as an international standard to protect against interplanetary biological…

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normalization of ChIP-seq data by using the spike-ins or by using total library sizes

Dear all, This question may have been asked before, I have searched the mailing list and I can not find an answer. The question is about the correct way of setting the SizeFactors() in DESeq2 in 3 situations. I would like to double check with you. Although the R code…

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TAmiRNA NextGen Sequencing pipeline

Diagnostic innovator TAmiRNA now has in its product pipeline an innovative NGS technology for absolute quantitation of miRNAs and other small RNAs , the miND® (microRNA Next-Generation Sequencing Discovery) spike-in. The miND® spike-in has been developed for small RNA sequencing experiments and absolute quantitation of microRNAs in any biological matrix…

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what can cause high(> 80%) spike-ins and low read count (

what can cause high(> 80%) spike-ins and low read count (<100 reads) in low complexity ampseq run 1 Hello, I have data output from low complexity ampseq run. The read counts are very low (<100 reads per well) and the spike-ins% is very high (>80%) across all of the samples….

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Extracellular circulating miRNAs as stress-related signature to search and rescue dogs

Study approval was provided by the Research Ethics Committee of the University of Perugia (report n.2018-21 of 11/12/2018) according to Italian Ministry of Health legislation18. All methods were carried out following relevant guidelines and regulations and the study was carried out in compliance with the ARRIVE guidelines. Informed consent is…

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eli5 RNA Spike-ins : explainlikeimfive

I’m a data scientist trying to branch out into more bioinformatics/genetics. My tutorial is counting spike-ins, but his explanation was over my head: 1 – AnnData and Preprocessing spike-ins | Kaggle ” Because this is smartseq2 data, we may have spike-ins. An RNA spike-in is an RNA transcript of known…

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Use Spike-Ins or TMM-normalization

Use Spike-Ins or TMM-normalization 1 Hi all, Sorry for all my questions lately, but as a novice which has to figure out how to analyse QuantSeq data, this forum has been a great and indispensible help for me. I’m doing a human transcriptomics analysis where we have QuantSeq data for…

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