KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples.

Jeremiah J Minich,John Hyde,Jae H Kim,Russ Vetter,Megan M Doty,Ryan Hendrickson,Kristina Stillwell,Kasthuri Venkateswaran,Stefan Janssen,Eric E Allen,Qiyun Zhu,Rob Knight,James Benardini,Amnon Amir

doi:10.1128/msystems.00218-17

Abstract

Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ low-throughput methods with minimal controls. We developed a new automated protocol, KatharoSeq (from the Greek katharos [clean]), that outperforms single-tube extractions while processing at least five times as fast. KatharoSeq incorporates positive and negative controls to reveal the whole bacterial community from inputs of as few as 50 cells and correctly identifies 90.6% (standard error, 0.013%) of the reads from 500 cells. To demonstrate the broad utility of KatharoSeq, we performed 16S rRNA amplicon and shotgun metagenome analyses of the Jet Propulsion Laboratory spacecraft assembly facility (SAF; n = 192, 96), 52 rooms of a neonatal intensive care unit (NICU; n = 388, 337), and an endangered-abalone-rearing facility (n = 192, 123), obtaining spatially resolved, unique microbiomes reproducible across hundreds of samples. The SAF, our primary focus, contains 32 sOTUs (sub-OTUs, defined as exact sequence matches) and their inferred variants identified by the deblur algorithm, with four (Acinetobacter lwoffii, Paracoccus marcusii, Mycobacterium sp., and Novosphingobium) being present in >75% of the samples. According to microbial spatial topography, the most abundant cleanroom contaminant, A.lwoffii, is related to human foot traffic exposure. In the NICU, we have been able to discriminate environmental exposure related to patient infectious disease, and in the abalone facility, we show that microbial communities reflect the marine environment rather than human input. Consequently, we demonstrate the feasibility and utility of large-scale, low-biomass metagenomic analyses using the KatharoSeq protocol. IMPORTANCE Various indoor, outdoor, and host-associated environments contain small quantities of microbial biomass and represent a niche that is often understudied because of technical constraints. Many studies that attempt to evaluate these low-biomass microbiome samples are riddled with erroneous results that are typically false positive signals obtained during the sampling process. We have investigated various low-biomass kits and methods to determine the limit of detection of these pipelines. Here we present KatharoSeq, a high-throughput protocol combining laboratory and bioinformatic methods that can differentiate a true positive signal in samples with as few as 50 to 500 cells. We demonstrate the application of this method in three unique low-biomass environments, including a SAF, a hospital NICU, and an abalone-rearing facility.

Highlights

Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ lowthroughput methods with minimal controls
Spacecraft assembly facilities (SAFs) are extremely low-microbial-biomass environments, even compared to other built or low-biomass environments such as hospitals [6], pharmaceutical production facilities [8], and indoor environments [3], because the National Aeronautics and Space Administration (NASA) takes extreme steps to avoid the transfer of any terrestrial contaminants to other planets [9]
With a 50-cell input, we could differentiate the positive control, B. subtilis, from the negative controls, only 28.8% of the sequences aligned with the target

Summary

Introduction

Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ lowthroughput methods with minimal controls. To demonstrate the broad utility of KatharoSeq, we performed 16S rRNA amplicon and shotgun metagenome analyses of the Jet Propulsion Laboratory spacecraft assembly facility (SAF; n ϭ 192, 96), 52 rooms of a neonatal intensive care unit (NICU; n ϭ 388, 337), and an endangered-abalone-rearing facility (n ϭ 192, 123), obtaining spatially resolved, unique microbiomes reproducible across hundreds of samples. KatharoSeq enables high-throughput microbiome analysis from low-biomass JPL spacecraft assembly facility, NICU, and abalone-rearing facility msystems.asm.org 1 environment, low biomass, metagenomics, microbial ecology, neonatal intensive care unit, NICU. More than 15 years of microbiological surveys of various NASA and European Space Agency cleanroom facilities showed that 1 to 10 m2 needs to be sampled to obtain reproducible microbiome signatures by Sanger sequencing, PhyloChip, 454 pyrosequencing, or Illumina sequencing [2] These procedures are critical because NASA allows only swabs, not larger sampling devices, for the collection of materials from sensitive spacecraft hardware surfaces (e.g., components of a sampling or life detection system, typically with surface areas of Ͻ1 m2). Many other low-microbial-biomass environments are of considerable interest, including neonatal intensive care unit (NICU) [10, 11] and aquaculture [12] settings, motivating the development of a general technique that works across these environments

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