title: 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.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Tourlousse
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Satowa
authors.initials: S
authors.lastname: Yoshiike
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ohashi
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsukura
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics; D019295:Computational Biology; D004783:Environmental Microbiology; D059014:High-Throughput Nucleotide Sequencing; D054892:Metagenome; D056186:Metagenomics / Q000379:methods / Q000592:standards; D064307:Microbiota; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012015:Reference Standards
chemical_list: D012336:RNA, Ribosomal, 16S