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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Tourlousse DM,Yoshiike S,Ohashi A,Matsukura S,Noda N,Sekiguchi Y

doi

10.1093/nar/gkw984

subject

Has Abstract

pub_date

2017-02-28 00:00:00

pages

e23

issue

4

eissn

0305-1048

issn

1362-4962

pii

gkw984

journal_volume

45

pub_type

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