Application Note: Microbial Genomics

16S Metagenomics Studies with the MiSeq® System
From DNA isolation to analysis, this simple workflow enables species-level identification ofcomplex microbial populations in 2 days.
Introduction

Figure 1: Multiplexed 16S Amplicon
Sequencing Workflow
Custom Probe 2Metagenomic surveys of microbial populations are often performed
using the prokaryotic 16S ribosomal RNA (rRNA) gene, which contains
conserved and variable regions that facilitate sequencing andphylogenetic classification. Following the complete Illumina workflow(Figure 1), 16S metagenomics studies with the MiSeq System canachieve species-level identification of microbial populations efficiently.
The workflow includes DNA isolation, library preparation, sequencing,and push-button analysis, delivering an end-to-end solution for 16S
metagenomics. By combining the demonstrated Illumina library
preparation protocol, the MiSeq System, and simple analysis software,
researchers can analyze complex microbial samples quickly and easily.

Custom Probe 1

Design and order region of interest-specificprimers with overhang adapters.

Generate template libraries and attach indexes
and sequencing adapters by PCR.

This application note provides an overview of the Illumina 16S
metagenomics workflow and the results of a study that examined
microbial populations in a water reservoir. This study was conducted incollaboration with the Center for Earth and Environmental Science at
Indiana University–Purdue University Indianapolis (IUPUI) and Citizens
Energy Group. The results reveal patterns in the reservoir’s microbialcommunity that can potentially be used to assess environmental
influences on water quality.

Perform automated cluster generation andsequencing on the MiSeq System.

OthGammerSample Collection
Water samples were collected from the Eagle Creek reservoir inIndianapolis, Indiana at regular intervals throughout one year. Discretesamples were collected from the surface and at depths of 3 meters,
6 meters, and near the bottom floor of the reservoir.

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The study used the 16S Metagenomic Sequencing Library Preparation
Guide1 to prepare sequencing libraries targeting the variable V3and V4 regions of the 16S rRNA gene. Paired-end sequencing wasperformed on the MiSeq System and data were analyzed using the 16S
Metagenomics App in the BaseSpace® analysis environment.

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Workflow in MiSeq Reporter software or the 16S
Metagenomics App in BaseSpace.

From DNA isolation to push-button analysis, the Illumina 16S
metagenomics workflow provides a complete solution for complex
community analyses. Sequencing with the MiSeq System delivers highly
accurate data, and analysis includes classification using BaseSpace or
MiSeq Reporter software.

Table 1: DNA Isolation Kits

DNA Isolation
Epicentre® DNA isolation kits deliver high-quality, inhibitor-free DNA
from mixed samples of gram-positive and gram-negative bacteria
derived from many environmental sources, including water, soil, fecal
matter, and compost (Table 1). In this study, DNA was isolated from
27 water samples using the Meta-G-NomeTM DNA Isolation Kit2.
Approximately 700 ng of DNA were extracted from each sample.

Sample TypeIsolation Kit

Water

Metagenomic DNA
Isolation Kit for Water3Soil

SoilMasterTM DNA
Extraction Kit4

Fecal matter
Difficult-to-culture species present inenvironmental water, soil, or compost

ExtractMasterTM Fecal
DNA Extraction Kit5
Meta-G-Nome
DNA Isolation Kit

Epicentre DNA isolation kits are optimized to isolate bacterial DNA obtained
from various environments.

Application Note: Microbial Genomics
Figure 2: 16S Metagenomics App

Table 2: MiSeq System Configurations

No. ofReads

ReadLength

Output600-cycle V3
standard flow cell

25 M

2 × 300 bp

15 Gb

500-cycle V2
standard flow cell

15 M

2 × 250 bp8 Gb

300-cycle V2
micro flow cell

4M

2 × 150 bp

1.2 Gb500-cycle V2nano flow cell

1M

2 × 250 bp

0.5 GbFlow Cell

No. of 16SSamples
Per RunHundreds of16S samples

Tens of 16Ssamples

The 16S Metagenomics App features an intuitive user interface to simplify
analysis of environmental samples.

Figure 3: Relative Abundance by Taxonomic Level

Library Preparation
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The MiSeq System can deliver 2 × 300 bp reads and up to50 million paired-end reads, generating up to 15 Gb of data. Theflexible system enables microbiologists to scale studies from one
to hundreds of samples. Micro and nano flow cell options andaccompanying reagents are available to support lower-throughput
experiments by optimizing sample volume and coverage needs(Table 2).
Samples from the reservoir were loaded onto a MiSeq reagent
cartridge and then onto the instrument. Automated cluster generation
and a 2 × 300 bp paired-end sequencing run were performed. The
resulting sequence reads were equally distributed across the samples,
demonstrating uniform coverage.

Data Analysis
Illumina has removed much of the complexity from sequencing
data analysis. Following the Illumina workflow, researchers cananalyze sequencing data generated on the MiSeq System either on
the instrument or in BaseSpace. MiSeq Reporter software is able
to analyze data on the sequencer or on a standalone computer.
Alternatively, data can be transferred, analyzed, stored, and sharedwith collaborators in BaseSpace. BaseSpace can deliver analyzed
sequences in as little as 12 hours following the 16S workflow, andBaseSpace applications (apps) provide access to a growing collectionof analysis tools.

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heBeThe Illumina 16S Metagenomic Sequencing Library Preparation Guideis an easy-to-follow protocol for preparing DNA libraries. It is optimized
to target the V3 and V4 regions of the 16S rRNA gene, although it canbe adapted to target other variable regions. The 16S Metagenomic
Sequencing Library Preparation Guide leads users through eachstep of library preparation, from genomic DNA to sequencing-readylibraries. All necessary reagents are listed, including the required primer
sequences that target the V3 and V4 regions of the 16S rRNA gene.
These primers can also be modified to target different regions of the16S gene, or altered for custom applications. The 27 samples from thereservoir were prepared using the 16S library preparation protocol andthe Nextera® XT DNA Index Kit6 for cost-effective sample multiplexing.

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The interactive sunburst chart in the BaseSpace 16S Metagenomics App
shows the relative abundance of bacterial species. Users can select any
classification to magnify a taxonomic level of interest. This chart shows therelative abundance of species in a single sample from the Eagle Creek reservoir.

The reservoir samples were analyzed using the BaseSpace 16S
Metagenomics App (Figure 2). The app delivers all phylogenetic
data—including coverage statistics and detected species—in intuitive,easy-to-analyze reports. Sequencing reads are classified against theGreengenes7 database, achieving up to species-level sensitivity.

Results
The 16S Metagenomics App delivers highly interactive visualizations
for exploring taxonomic classifications. The sunburst classification
chart provides a detailed view of the relative abundance of bacterial
species within each taxonomic level. Researchers can select a
category to magnify a particular level of interest and explore thediversity of any sample (Figure 3).

Application Note: Microbial Genomics
Figure 4: Aggregate Species Diversity

The 16S Metagenomics App delivers a dendrogram that shows a hierarchical clustering of samples based on genus-level classifications. The bar charts show therelative abundance of the classifications present in each sample. Each bar color corresponds to a unique genus.

The 16S Metagenomics App also provides an aggregate summary
report so that researchers can compare the similarities and differencesamong samples within a project. The hierarchical dendrogram shows aclustering of samples based on genus-level classifications and therelative abundance of each (Figure 4). Detailed results and analysis fromthe Eagle Creek reservoir are available to view in BaseSpace8. Analysisof relative abundance in the reservoir revealed an increase in
Rhodococcus species in July. Various factors may have caused thesurge in Rhodococcus abundance, such as algaecide treatment or theuse of fertilizer near the reservoir. Further analysis is required to assess
the influences that contributed to changes in the community.

References1.

Illumina (2013) 16S Metagenomic Sequencing Library Preparation Guide.
(support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.ilmn)

2.

Meta-G-Nome DNA Isolation Kit (www.epibio.com/applications/nucleicacid-purification-extraction-kits/dna-purification-genomic/meta-g-nomedna-isolation-kit) Accessed 07 July 2014.

3.

Metagenomic DNA Isolation Kit for Water (www.epibio.com/applications/
nucleic-acid-purification-extraction-kits/dna-purification-genomic/metagenomic-dna-isolation-kit-for-water) Accessed 07 July 2014.

4.

SoilMaster- DNA Extraction Kit (www.epibio.com/applications/nucleic-acidpurification-extraction-kits/dna-extraction/soilmaster-dna-extraction-kit)
Accessed 07 July 2014.

5.

ExtractMaster- Fecal DNA Extraction Kit (www.epibio.com/applications/
nucleic-acid-purification-extraction-kits/dna-extraction/extractmaster-fecaldna-extraction-kit) Accessed 07 July 2014.

6.

Nextera XT DNA Library Prep Kit | Illumina (www.illumina.com/products/
nextera_xt_dna_sample_prep_kit.ilmn) Accessed 07 July 2014.

7.

DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, et al. (2006)
Greengenes, a chimera-checked 16S rRNA gene database and workbenchcompatible with ARB. Appl Environ Microbiol 72: 5069–72.

8.

Project - BaseSpace (basespace.illumina.com/projects/9954952/MiSeq-v3Nextera-XT-Metagenomics-Water-Samples) Accessed 07 July 2014.

Conclusions
Using the 16S metagenomics workflow with the MiSeq System,
microbiologists can achieve up to species-level sensitivity formetagenomic surveys of bacterial populations. In this study, theIllumina workflow was used to study microbial populations in areservoir, uncovering shifts in community composition. This researchenables biologists to investigate new methods for understanding
environmental influences on water sources and water quality. 16S
metagenomic studies comprise one of many applications empoweredby the MiSeq desktop sequencer. Illumina solutions support
researchers during every step of the process, from DNA isolation
through data analysis, enabling a range of applications for microbial
genomic discovery.

Learn More
For more information about the use of Illumina technology in microbial
genomics, visit www.illumina.com/microbiology. To learn more about
the MiSeq desktop sequencer, go to www.illumina.com/miseq.

Application Note: Microbial Genomics

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Illumina, BaseSpace, Epicentre, ExtractMaster, Meta-G-Nome, MiSeq, Nextera, SoilMaster, the pumpkin orange color, and thestreaming bases design are trademarks or registered trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or othercountries. All other names, logos, and other trademarks are the property of their respective owners.
Pub. No. 1270-2013-001 Current as of 17 October 2017