Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing

Abstract

High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.

Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows

“…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]. We built upon these studies to demonstrate the use of a spike-in for validating a shotgun/untargeted metagenomics workflow.…”

Casting a broader net: Using microfluidic metagenomics to capture aquatic biodiversity data from diverse taxonomic targets

“…These pathogens are the focus of pathogen surveys across the United States, as management of Phytophthora ‐infected zones seasonally impacts timber harvesting, recreation, and restoration activities (U.S. Department of the Interior, Bureau of Land Management, and U.S. Department of Agriculture, Forest Service ). Given their ubiquity, Phytophthora , Saprolegnia , or similarly cosmopolitan oomycetes may also provide “internal positive” controls that can be used in lieu of spike‐in controls (Tourlousse et al, ) for sample validation, or as a check for errors in collection (e.g., filtration) or DNA extraction methods. Our assay included primers for additional animal pathogens, specifically amphibian chytrid fungus ( Batrachochytrium dendrobatidis Longcore, Pessier & Nichols; Olson et al, ) and the fungus that causes white‐nose syndrome in bats ( Pseudogymnoascus destructans (Blehert & Gargas) Minnis & Lindner; Blehert et al, ).…”

“…de Bary), and other trees and shrubs (Hansen et al, 2012). Saprolegnia, or similarly cosmopolitan oomycetes may also provide "internal positive" controls that can be used in lieu of spike-in controls (Tourlousse et al, 2017) for sample validation, or as a check for errors in collection (e.g., filtration) or DNA extraction methods.…”

Microbiota in obesity: interactions with enteroendocrine, immune and central nervous systems

“…Second, the HFD‐induced changes in microbiota are often inconsistent, likely due to differences in the experimental approaches. This can be different, for example, depending on (i) whether the microbiota was analysed in the faeces or in the intestinal contents such as the caecum; (ii) different methodologies used for sample processing such as DNA extraction or sequencing ; (iii) the duration of the intervention with a HFD ; (iv) the source of dietary fat ; (v) the concentration of sucrose in the diet and (vi) the initial microbiota composition and the genetic background of the animals . Additionally, a recent study found that mice from the same strain, which consumed the same diet, but reside in different facilities within Germany, had significantly different faecal microbiota .…”