Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis

Abstract

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed “sequins” (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as reference standards to aid in metagenomic studies.

The quest for absolute abundance: the use of internal standards for DNA-based microbial and community ecology

“…"A method for simultaneous measurement of soil bacterial abundances and community composition via 16S rRNA gene sequencing". Hardwick et al (2018). "Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis".…”

“…These researchers 259 suggest simply reversing the portion of the genome of the focal taxon under consideration 260 (e.g., the portion of the rRNA operon commonly used for molecular metabarcoding). The 261 approach ofHardwick et al 2018 was suggested for shotgun metagenomics. Notably, if such 262 a technique is used for single-locus, metabarcoding, correct-sense primer sequences must be 263 appended to the reversed sequence to ensure amplification.…”

A Quantitative Sequencing Framework for Absolute Abundance Measurements of Mucosal and Lumenal Microbial Communities

“…Spiked standards are commonly used in method calibration and have recently been applied to quantifying taxa in microbiome research. [19][20][21][22][23] These methods require a purified DNA sequence of known concentration from an organism not present in the sample and an estimate of the initial sample concentration to determine the amount of exogenous DNA to spike-in. Another group of anchoring methods, such as those that use flow cytometry 24 , total DNA 25 , or qPCR [26][27][28] , measure the total concentration of cells, DNA, or amplicons to transform the relative abundances to absolute numbers.…”

Identifying accurate metagenome and amplicon software via a meta-analysis of sequence to taxonomy benchmarking studies

“…The accuracy of the genome assembly, genome partitioning (binning) and read depth proportional to consortium makeup can then be used to confirm software accuracy. In principle, every metagenome experiment could employ in vitro positive and negative controls by "spiking" known amounts of DNA from known sources, as has been widely used for gene expression analysis [44] and increasingly for metagenomics [45][46][47] . In silico methods use selected publicly-available genome sequences.…”