The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses

Chen, Kaifu; Hu, Zheng; Xia, Zheng; Zhao, Dongyu; Li, Wei; Tyler, Jessica K.

doi:10.1128/mcb.00970-14

Cited by 173 publications

(164 citation statements)

References 10 publications

Supporting

Mentioning

163

Contrasting

Order By: Relevance

“…The use of a spike-in approach for QC in sequencing RNA and DNA has a long history and its importance has been echoed in multiple publications [20,21]. Many recent publications have highlighted the use of the spike-in for 16S-rRNA encoding gene surveys, both for QC as well as a normalization tool to move from relative towards absolute abundance [13,22,23].…”

Section: Resultsmentioning

confidence: 99%

Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows

Venkataraman

Parlov

et al. 2018

BioTechniques

View full text Add to dashboard Cite

Shotgun metagenomics is a powerful platform to characterize human microbiomes. However, to translate such survey data into consumer-relevant products or services, it is critical to have a robust metagenomics workflow. We present a tool - spike-in DNA - to assess performance of metagenomics workflows. The spike-in is DNA from two organisms - Alivibrio fischeri and Rhodopseudomonas palustris, in a ratio of 4:1 added to samples before DNA extraction. With a valid workflow, the output ratio of relative abundances of these organisms should be close to 4. This expectation was tested in samples of varying diversities (n = 110), and the mean ratio was 4.73 (99% CI [4.0, 5.24]). We anticipate this tool to be a relevant community resource for assessing the quality of shotgun metagenomics workflows and thereby enable robust characterization of microbiomes.

show abstract

Section: Resultsmentioning

confidence: 99%

Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows

Venkataraman

Parlov

et al. 2018

BioTechniques

View full text Add to dashboard Cite

show abstract

“…Both methods use a set of common references for data normalization and apply an identical scaling approach based on some cumulative value for these common references [16]. Additionally, others have acknowledged that in the absence of spike-in controls, regions of the genome that have stable quantifiable occupancy through all experimental conditions are appropriate to use as an alternative for ChIP-seq data normalization [13, 16]. Here, due to our dataset lacking spike-in controls, we followed this strategy and, through qPCR validation, demonstrated it to indeed yield robust results.…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia

Adriaens

Prickaerts

Chan-Seng-Yue

et al. 2016

Epigenetics & Chromatin

View full text Add to dashboard Cite

BackgroundA comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets.ResultsWe first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells.Conclusions In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0090-4) contains supplementary material, which is available to authorized users.

show abstract

“…3 vs. 4). This could be overcome potentially through incorporation of spike-in controls at each round of the selection process (55). To date, we have applied this method to identify examples where protein partners exert substantial effects on the specificity and affinity of RNA-binding proteins (17, 18).…”

Section: Discussionmentioning

confidence: 99%

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)

Lou

Weidmann

Killingsworth

et al. 2017

Methods

View full text Add to dashboard Cite

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA.

show abstract

The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses

Cited by 173 publications

References 10 publications

Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows

Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)

Contact Info

Product

Resources

About