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Microbiome
Analysis
Robert G. Beiko
Will Hsiao
John Parkinson Editors
Methods and Protocols
Methods in
Molecular Biology 1849

M E T H O D S I N M O L E C U L A R B I O L O G Y
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Microbiome Analysis
Methods and Protocols
Edited by
Robert G. Beiko
Faculty of Computer Science, Dalhousie University, Halifax, NS, Canada
Will Hsiao
Department of Pathology & Laboratory Medicine, University of British Columbia,
Vancouver, BC, Canada
John Parkinson
Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON, Canada;
Department of Biochemistry and Department of Molecular Genetics, University of Toronto,
Toronto, ON, Canada

Editors
Robert G. Beiko
Faculty of Computer Science
Dalhousie University
Halifax, NS, Canada
Will Hsiao
Department of Pathology
& Laboratory Medicine
University of British Columbia
Vancouver, BC, Canada
John Parkinson
Program in Molecular Medicine
The Hospital for Sick Children
Toronto, ON, Canada
Department of Biochemistry
and Department of Molecular Genetics
University of Toronto
Toronto, ON, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-8726-9 ISBN 978-1-4939-8728-3 (eBook)
https://doi.org/10.1007/978-1-4939-8728-3
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Preface
The microbiome, coined by Lederberg and McCray as “...the ecological community of
commensal, symbiotic, and pathogenic microorganisms that literally share our body space”
[1], draws together a remarkable number of disciplines with the overarching goal of
understanding and ultimately harnessing the workings of microbial systems. True to the
initial conception of the term, the human microbiome continues to be intensively studied,
but microbial samples have been collected from nearly every imaginable habitat on Earth,
from the upper atmosphere to the seabed subsurface, from hot springs to glacier ice, and
from nematode guts to whale carcasses.
Microbiome analysis makes frequent use of a common set of tools to address many
pertinent questions. A common workflow for microbiome analysis looks something like this:
collect sample (e.g., soil, water, stool), extract DNA, perform DNA sequencing, and use
bioinformatics tools to describe important properties of the microbiome. This pipeline has
been applied to huge numbers of samples from a diverse array of environments. In particular,
the targeted sequencing via polymerase chain reaction (PCR) amplification of “marker”
genes that are seen as diagnostic of different types of microorganisms has seen widespread
use. The workhorse of microbial diversity has thus far been the 16S ribosomal RNA gene,
which has been the subject of intensive protocol development: see for example the Earth
Microbiome Project protocols [2], and a detailed evaluation of 16S sequencing on the
Illumina sequencing platforms [3]. However, while capturing the taxonomic composition of a
microbial community, marker-gene sequencing is limited in its ability to reveal the diversity
of functions present, requiring the application of alternative approaches.
Gaining an accurate and relevant picture of the microbiome requires careful experimen-
tal design, and the first part of our book focuses on the profiling of different habitats and
elements of biodiversity. The procedures to collect representative and uncontaminated
samples can be highly complex; one need look no further than Chapter 1 for an example
of the challenges associated with collecting microbial samples from the deep subsurface.
DNA sequencing might be seen as the foundation of microbial community analysis, even if
arguably the first such analysis was done with RNA rather than DNA sequencing in the
famous Octopus Spring study [4]. However, other “meta-omic” methods that consider
messenger RNA transcripts, protein products, or metabolite levels can reveal a great deal
more about microbial activities in a particular habitat. The combination of multiple such
strategies can be especially powerful, as illustrated by the use of DNA sequencing to support
targeted metaproteomics (Chapter 6).
The remainder of this volume addresses the computational challenges of microbiome
analysis. An immense number of algorithms and software packages have been developed for
the task, and even seemingly simple questions as “what is the biodiversity present in a given
sample?” may not be straightforward, as exemplified by Chapter 10. At the same time, the
rich information generated from these samples is driving the development of innovative
tools and pipelines with the ability to generate novel data types and address new questions,
such as the recovery of complete genomes from metagenomes (Chapter 14), and the use of
network approaches to identify patterns of microbial association (Chapter 17).
Although no book on microbiome analysis can be exhaustive, in preparing our volume
we have sought to convey what might be seen as standard practice in the field (to the extent
v

anything can be claimed as such!), while also highlighting techniques at the frontiers of the
field that challenge standard practice. Reflecting the continued dominance of marker-gene
approaches, the QIIME package [5] recently received its ten-thousandth citation: the recent
release of the second version of this software is notable because it is developed in a
completely different framework, and because it upends some of the tools and techniques
that have heretofore served as its defaults (see Chapter 8).
By defining procedures in precise terms, the Methods in Molecular Biology series con-
tributes to the reproducibility of experiments. However, reproducibility in bioinformatics is
a big concern [6], with several moving parts including database versions, software updates,
and parameter settings. Comparing new methods to existing ones demands that final results
and all intermediate steps can be regenerated. The last few years have seen significant
advances in reproducibility through means such as automated workflow tools including
Galaxy, interactive code tools such as Jupyter Notebooks, and repositories with version
control, the most notable example of which is currently Github. We are pleased that many of
our authors have provided examples that make use of these tools, which will make it
considerably easier for readers to perform analyses in a consistent manner.
It remains only for us to thank the individuals who have contributed their time and hard
work to preparing a highly diverse and engaging set of chapters. We are also grateful to John
Walker for the original invitation to prepare this book.
Halifax, NS, Canada Robert G. Beiko
Vancouver, BC, Canada Will Hsiao
Toronto, ON, Canada John Parkinson
References
1. Lederberg J, McCray AT (2001) Ome SweetOmics—a genealogical treasury of words.
Scientist 15;8
2. Earth Microbiome Project. Protocols and Standards. http:/
/www.earthmicrobiome.
org/protocols-and-standards/. Accessed 3 March 2018
3. Caporaso JG, Lauber CL, Walters WA, et al (2012) Ultra-high-throughput microbial
community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621
4. Stahl DA, Lane DJ, Olsen GJ, et al (1985) Characterization of a Yellowstone hot spring
microbial community by 5S rRNA sequences. Appl Environ Microbiol 49:1379–1384
5. Caporaso JG, Kuczynski J, Stombaugh J et al (2010). QIIME allows analysis of high-
throughput community sequencing data. Nat Methods 7:335
6. Sandve GK, Nekrutenko A, Taylor J et al (2013) Ten simple rules for reproducible
computational research. PLoS Comput Biol 9:e1003285
vi Preface

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Characterizing the Deep Terrestrial Subsurface Microbiome . . . . . . . . . . . . . . . . . 1
Rebecca A. Daly, Kelly C. Wrighton, and Michael J. Wilkins
2 Freshwater Viromes: From Sampling to Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . 17
Catherine Putonti, Zoë Diener, and Siobhan C. Watkins
3 Characterization of Eukaryotic Microbiome Using 18S
Amplicon Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Ana Popovic and John Parkinson
4 Culture and Molecular Profiling of the Respiratory Tract Microbiota. . . . . . . . . . 49
Fiona J. Whelan, Laura Rossi, Jennifer C. Stearns,
and Michael G. Surette
5 Methods and Strategies to Examine the Human Breastmilk Microbiome . . . . . . 63
Lauren LeMay-Nedjelski, Julia Copeland, Pauline W. Wang,
James Butcher, Sharon Unger, Alain Stintzi, and Deborah L. O’Connor
6 Quantification of Vitamin B12-Related Proteins in Marine
Microbial Systems Using Selected Reaction Monitoring
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Erin M. Bertrand
7 Single-Cell Genomics of Microbial Dark Matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Christian Rinke
8 16S rRNA Gene Analysis with QIIME2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Michael Hall and Robert G. Beiko
9 Processing a 16S rRNA Sequencing Dataset with the Microbiome
Helper Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Gavin M. Douglas, André M. Comeau, and Morgan G. I. Langille
10 Normalization of Microbiome Profiling Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Paul J. McMurdie
11 Predicting the Functional Potential of the Microbiome
from Marker Genes Using PICRUSt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Gavin M. Douglas, Robert G. Beiko, and Morgan G. I. Langille
12 Metagenome Assembly and Contig Assignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Qingpeng Zhang
13 From RNA-seq to Biological Inference: Using Compositional
Data Analysis in Meta-Transcriptomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Jean M. Macklaim and Gregory B. Gloor
14 Subsampled Assemblies and Hybrid Nucleotide
Composition/Differential Coverage Binning for Genome-Resolved
Metagenomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Laura A. Hug
vii

15 Transkingdom Networks: A Systems Biology Approach to Identify
Causal Members of Host–Microbiota Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Richard R. Rodrigues, Natalia Shulzhenko, and Andrey Morgun
16 Constructing and Analyzing Microbiome Networks in R . . . . . . . . . . . . . . . . . . . . 243
Mehdi Layeghifard, David M. Hwang, and David S. Guttman
17 Bayesian Inference of Microbial Community Structure
from Metagenomic Data Using BioMiCo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Katherine A. Dunn, Katelyn Andrews, Rana O. Bashwih,
and Joseph P. Bielawski
18 Analyzing Metabolic Pathways in Microbiomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Mobolaji Adeolu, John Parkinson, and Xuejian Xiong
19 Sparse Treatment-Effect Model for Taxon Identification
with High-Dimensional Metagenomic Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Zhenqiu Liu and Shili Lin
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
viii Contents

Contributors
MOBOLAJI ADEOLU Program in Molecular Medicine, The Hospital for Sick Children,
Toronto, ON, Canada
KATELYN ANDREWS Department of Mathematics and Statistics, Dalhousie University,
Halifax, NS, Canada
RANA O. BASHWIH Department of Mathematics and Statistics, Dalhousie University,
Halifax, NS, Canada
ROBERT G. BEIKO Faculty of Computer Science, Dalhousie University, Halifax, NS,
Canada
ERIN M. BERTRAND Department of Biology, Dalhousie University, Halifax, NS, Canada
JOSEPH P. BIELAWSKI Department of Mathematics and Statistics, Dalhousie University,
Halifax, NS, Canada
JAMES BUTCHER Ottawa Institute of Systems Biology, Ottawa, ON, Canada; Department of
Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
ANDRÉ M. COMEAU Integrated Microbiome Resource, Dalhousie University, Halifax, NS,
Canada
JULIA COPELAND Centre for the Analysis of Genome Evolution and Function, University of
Toronto, Toronto, ON, Canada
REBECCA A. DALY Department of Microbiology, The Ohio State University, Columbus, OH,
USA
ZOË DIENER Department of Biology, New Mexico Institute for Mining and Technology,
Socorro, NM, USA
GAVIN M. DOUGLAS Department of Microbiology and Immunology, Dalhousie University,
Halifax, NS, Canada
KATHERINE A. DUNN Department of Mathematics and Statistics, Dalhousie University,
Halifax, NS, Canada
GREGORY B. GLOOR Department of Biochemistry, Schulich School of Medicine and
Dentistry, The University of Western Ontario, London, ON, Canada; Canadian Centre
for Human Microbiome and Probiotic Research, Lawson Health Sciences Centre, London,
ON, Canada
DAVID S. GUTTMAN Department of Cell and Systems Biology, University of Toronto, Toronto,
ON, Canada; Centre for the Analysis of Genome Evolution and Function, University of
MICHAEL HALL Dalhousie University, Halifax, NS, Canada
LAURA A. HUG Department of Biology, University of Waterloo, Waterloo, ON, Canada
DAVID M. HWANG Department of Pathology, University Health Network, Toronto, ON,
Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, ON, Canada
MORGAN G. I. LANGILLE Department of Microbiology and Immunology, Dalhousie
University, Halifax, NS, Canada; Integrated Microbiome Resource, Dalhousie University,
Halifax, NS, Canada; Department of Pharmacology, Dalhousie University, Halifax, NS,
Canada
MEHDI LAYEGHIFARD Department of Cell and Systems Biology, University of Toronto,
Toronto, ON, Canada
ix

LAUREN LEMAY-NEDJELSKI Faculty of Medicine, Department of Nutritional Sciences,
University of Toronto, Toronto, ON, Canada; Translational Medicine, The Hospital for
Sick Children, Toronto, ON, Canada
SHILI LIN Department of Statistics, The Ohio State University, Columbus, OH, USA
ZHENQIU LIU Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical
Center, Los Angeles, CA, USA
JEAN M. MACKLAIM Department of Biochemistry, Schulich School of Medicine and Dentistry,
The University of Western Ontario, London, ON, Canada; Canadian Centre for Human
Microbiome and Probiotic Research, Lawson Health Sciences Centre, London, ON, Canada
PAUL J. MCMURDIE Whole Biome, Inc., San Francisco, CA, USA
ANDREY MORGUN College of Pharmacy, Oregon State University, Corvallis, OR, USA
DEBORAH L. O’CONNOR Faculty of Medicine, Department of Nutritional Sciences,
University of Toronto, Toronto, ON, Canada; Translational Medicine, The Hospital for
Sick Children, Toronto, ON, Canada; Department of Pediatrics, Mount Sinai Hospital,
Toronto, ON, Canada
JOHN PARKINSON Program in Molecular Medicine, The Hospital for Sick Children, Toronto,
ON, Canada; Department of Biochemistry and Department of Molecular Genetics,
University of Toronto, Toronto, ON, Canada
ANA POPOVIC Program in Molecular Medicine, The Hospital for Sick Children, Toronto,
ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, Canada
CATHERINE PUTONTI Department of Biology, Loyola University Chicago, Chicago, IL, USA;
Department of Computer Science, Loyola University Chicago, Chicago, IL, USA;
Bioinformatics Program, Loyola University Chicago, Chicago, IL, USA; Department of
Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago,
Maywood, IL, USA
CHRISTIAN RINKE Australian Centre for Ecogenomics, University of Queensland, Brisbane,
QLD, Australia
RICHARD R. RODRIGUES College of Pharmacy, Oregon State University, Corvallis, OR, USA
LAURA ROSSI Department of Biochemistry and Biomedical Sciences, McMaster University,
Hamilton, ON, Canada
NATALIA SHULZHENKO College of Veterinary Medicine, Oregon State University, Corvallis,
OR, USA
JENNIFER C. STEARNS Department of Medicine, McMaster University, Hamilton, ON,
Canada
ALAIN STINTZI Ottawa Institute of Systems Biology, Ottawa, ON, Canada; Department of
Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
MICHAEL G. SURETTE Department of Medicine, McMaster University, Hamilton, ON,
Canada
SHARON UNGER Translational Medicine, The Hospital for Sick Children, Toronto, ON,
Canada; Faculty of Medicine, Department of Pediatrics, University of Toronto, Toronto,
ON, Canada; Department of Pediatrics, Mount Sinai Hospital, Toronto, ON, Canada
PAULINE W. WANG Centre for the Analysis of Genome Evolution and Function, University of
SIOBHAN C. WATKINS Department of Biology, New Mexico Institute for Mining and
Technology, Socorro, NM, USA
FIONA J. WHELAN Department of Biochemistry and Biomedical Sciences, McMaster
University, Hamilton, ON, Canada
x Contributors

MICHAEL J. WILKINS Department of Microbiology, The Ohio State University, Columbus,
OH, USA
KELLY C. WRIGHTON Department of Microbiology, The Ohio State University, Columbus,
OH, USA
XUEJIAN XIONG Program in Molecular Medicine, The Hospital for Sick Children, Toronto,
ON, Canada
QINGPENG ZHANG Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA
Contributors xi

Chapter 1
Characterizing the Deep Terrestrial Subsurface Microbiome
Rebecca A. Daly, Kelly C. Wrighton, and Michael J. Wilkins
Abstract
A large portion of the earth’s biomass resides in the subsurface and recent studies have expanded our
knowledge of indigenous microbial life. Advances in the field of metagenomics now allow analysis of
microbial communities from low-biomass samples such as deep (2.5 km) shale core samples. Here we
present protocols for the best practices in contamination control, handling core material, extraction of
nucleic acids, and low-input library preparation for subsequent metagenomic sequencing.
Key words Deep life, Deep biosphere, Terrestrial subsurface, Contamination, Shale, Metagenomics,
Low biomass
1 Introduction
While it is estimated that the terrestrial subsurface is the largest
reservoir of life on Earth, hosting between 40% and 60% of all
bacterial cells, densities are typically low in deep subsurface ecosys-
tems [1, 2]. Research into the deep terrestrial biosphere stems from
interest in biogeochemical cycling and the discovery of novel bio-
diversity and metabolisms [3]. However, due to the difficulty in
obtaining core samples from thousands of meters below the Earth’s
surface and characteristic low biomass, it is critical that samples are
collected and preserved in a manner to limit contamination.
Advances in metagenomic sequencing technology which permits
library construction from picogram quantities of DNA can now be
utilized to examine not only the presence of single-gene markers
(i.e., 16S rRNA genes), but allow for reconstruction of entire
microbial genomes, providing insight into functional potential
within the deep terrestrial subsurface microbiome.
There is a large body of published work detailing how to deal
with contamination of low-biomass samples during drilling and
coring, and the sources of contamination [3–6]. Small amounts of
contaminating bacteria can mask the signal from indigenous
Robert G. Beiko et al. (eds.), Microbiome Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 1849,
https://doi.org/10.1007/978-1-4939-8728-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018
1

microorganisms and compromise future analyses. Methods often
include the use of tracers during the drilling process, including
microbial tracers (e.g., live cells) [7], chemical tracers such as per-
fluorocarbons [8], and visual tracers such as fluorescein [9] and
fluorescent microspheres [4, 10–13]. The collection of “blanks” at
multiple points of sample processing provides the ability to distin-
guish contaminating microorganisms from indigenous life. Once
contamination controls have been implemented, the question still
remains of how to extract DNA from low-biomass rock matrices.
DNA is commonly extracted by using a combination of chemi-
cal and physical lysis [14–18] in order to recover and subsequently
purify DNA from lysed cells, usable for downstream molecular
assays. There are a wide range of DNA-extraction protocols, with
certain methods optimized for particular sample types, including
commercial kits. For challenging and unique samples, there may
be no established methods. There is no “one-size-fits-all” DNA-
extraction protocol, and it is recommended that multiple methods
are tested and compared on sample material physically and chemi-
cally similar to the targeted samples. Once DNA has been success-
fully extracted and purified from the matrix, commercial kits are
available for low-input library preparation for metagenomic
sequencing [19, 20].
This chapter outlines procedures for contamination control in
the field, the use of fluorescent microsphere tracers during drilling,
contamination control in the lab, sample decontamination/clean-
ing, sample grinding, DNA extraction and library preparation for
metagenomic sequencing for core samples obtained from the Mar-
cellus shale formation in West Virginia, USA.
2 Materials
2.1 Collection
and DNA Extraction
of Drilling Muds
and Other Fluids
1. Nalgene 1 L wide-mouth HDPE bottles, autoclaved.
2. 0.2 μm polyethersulfone (PES) vacuum filter device.
3. MoBio PowerMax Soil DNA isolation kit (MoBio Labora-
tories, Carlsbad, CA, USA).
4. Standard PCR reagents and primers for 16S rRNA gene
amplification.
2.2 Fluorescent
Microspheres
1. Fluoresbrite YG carboxylate microspheres 0.50 μm (Poly-
sciences, Warrington, PA, USA).
2. Sterile carboy or other similar container filled with deionized
water with a sensitivity of 18 MΩ cm at 25
C.
2 Rebecca A. Daly et al.

2.3 Core Collection 1. Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) of the appro-
priate size to hold a single core.
2. Anaeropak 7.0 L rectangular jars (Mitsubishi, Tokyo, Japan).
3. AnaeroPouch System-Anaero Anaerobic Gas Generator sachets
(Mitsubishi, Tokyo, Japan).
2.4 Quantification
of Microspheres
1. Inverted, fluorescent microscope with a 10 objective (for
100 total magnification). Ensure that the microscope has
enough clearance to allow the core sample to be placed on
the stage. We used an Eclipse Ti inverted microscope (Nikon,
Tokyo, Japan).
2. Software for obtaining color micrographs.
2.5 Core Sample
Decontamination
1. 1.5 M NaCl solution: 87.66 g of NaCl. Add DNA-free water to
a volume of 1 L. Mix and autoclave.
2. Autoclaved aluminum dish with fluted sides, 200 mL (vol-
ume dependent on the size of core samples).
3. Autoclaved high quality, fine (size 00) steel wool. Autoclaved
steel wool wrapped in loose aluminum foil. After autoclaving
dry in a 50
C oven to prevent rusting.
2.6 Grinding of Core
Samples
1. Bunsen burner.
2. Metal tongs for holding core samples.
3. Autoclaved 12 12” sheets of aluminum foil.
4. Cleaned, autoclaved ceramic mortars and pestles.
5. Brass mesh sieves with SS wire, including sieve cover and
bottom (e.g., stackable 3” sieves in #10, #18, and #35 mesh
(Humboldt Manufacturing Company, Elgin, IL, USA)).
6. Plattner’s hardened steel mortars and pestles (Humboldt
Manufacturing Company, Elgin, IL, USA).
7. Cold chisel, ½” cut width.
8. Sledge/drilling hammer with compact handle (3 lb.
hammer).
2.7 DNA Extraction
from Ground Core
1. DNAZap PCR DNA Degradation solutions (ThermoFisher
Scientific, Waltham, MA, USA).
2. Lysis Buffer I, pH 10, 250 mL: Add 17.5 mL of 1.0 M, pH 7.5
Tris–HCl; 15.0 mL of 0.5 M, pH 8.0 EDTA, 25.0 mL of 8.0 M
guanidine hydrochloride, and 1.25 mL of Triton X-100 to a
sterile flask. Add DNA-free water to a volume of 250 mL. Mix
and adjust pH to 10.0 with NaOH. Filter sterilize through a
250 mL, 0.1 μm, PES vacuum filter unit.
3. DNA LoBind 1.5 mL tubes (Eppendorf, Hauppauge, NY,
USA).
Deep Terrestrial Subsurface 3

4. Ultra-high-speed 50 mL centrifuge tubes (VWR International,
Radnor, PA, USA).
5. Phenol:chloroform:isoamyl alcohol, 25:24:1, pH 8.0.
6. Chloroform:isoamyl alcohol, 24:1.
7. 100% ethanol.
8. 70% ethanol, prepared with DNA-free water.
9. EB buffer (Qiagen, Valencia, CA, USA).
10. Linear acrylamide, 5 mg/mL (ThermoFisher Scientific, Wal-
tham, MA, USA).
2.8 DNA
Quantification
1. Qubit fluorometer (ThermoFisher Scientific, Waltham, MA,
USA).
2. Qubit dsDNA HS (high sensitivity) assay kit (ThermoFisher
Scientific, Waltham, MA, USA).
3. Qubit assay tubes, 0.5 mL (ThermoFisher Scientific, Waltham,
MA, USA).
2.9 Library
Preparation
for Metagenomic
Sequencing
and Optional MDA
Amplification
1. REPLI-g Single Cell WGA kit (Qiagen, Valencia, CA, USA).
2. Nextera XT DNA library preparation kit (Illumina, Inc., San
Diego, CA, USA).
3. Nextera XT Index kit (Illumina, Inc., San Diego, CA, USA).
4. Agencourt AMPure XP magnetic beads (Beckman Coulter Life
Sciences, Indianapolis, IN, USA).
5. Magnetic stand for 1.5 mL microcentrifuge tubes.
6. Bioanalyzer Instrument (Agilent Technologies, Santa Clara,
CA, USA).
7. Bioanalyzer High Sensitivity DNA kit (Agilent Technologies,
Santa Clara, CA, USA).
3 Methods
3.1 Contamination
Control in the Field
Recovering core material from the subsurface requires drilling
technologies which can introduce contamination from several
sources, including drilling mud additives, surface water mixed
with drilling muds, contamination from mud tanks, pumping
equipment, and contamination from overlying formations and
groundwater. In order to obtain reliable information about indige-
nous microorganisms, it is extremely important to sample all
sources of potential contamination, extract DNA, and sequence
these samples for subtractive analysis. Chemical and particle tracers
are commonly used to assess sample integrity. Here we describe use
of fluorescent microspheres as a visual tracer, and DNA extraction
of the fluids used to clean core samples as a molecular tracer, to
ensure sample integrity.

3.1.1 Sampling Drilling
Muds and Other Fluids
1. Sample all fluids that can potentially go down-well, before and
during the drilling process starts, including freshwater added to
drilling muds, and the drilling muds (see Note 1).
2. Ensure that all sampling equipment is sterile, and wear dispos-
able gloves at all times. It is useful to have large ladles and
buckets on hand that are easily sterilized with a bleach solution
or 70% ethanol, for retrieving samples at the well pad.
3. Filter freshwater sources through a sterile, large-volume filter
apparatus with a 0.2 μm polyethersulfone (PES) membrane,
filtering a minimum of 3 L of each sample for future DNA
extraction and analysis. If the samples cannot be filtered in the
field, collect samples in sterile 1 L Nalgene containers, or larger
carboys, filled to the brim to minimize headspace and thus
alteration of the microbial community and transport on ice. If
the samples can be filtered in the field, have dry ice on hand to
rapidly freeze the filters during transport to the laboratory.
4. Collect drilling muds in 1 L sterile Nalgene containers.
5. DNA from freshwater source filters (5 g of filter material) and
aliquots of drilling muds (5 mL) can be extracted using the
manufacture’s recommended protocol using the PowerMax
Soil DNA isolation kit (see Note 2).
6. Test for PCR amplification using “universal” 16S rRNA gene
primers to ensure that samples are suitable for downstream
analyses (metagenomic library preparation).
3.1.2 Addition
of Fluorescent
Microspheres
to Drilling Muds
During the drilling process, drilling muds and other fluids can
penetrate core samples. We used Fluoresbrite carboxylate yellow-
green fluorescent polystyrene microspheres 0.50 μm in diameter as
a proxy for contaminating bacterial cells, and quantified micro-
sphere penetration and removal during the decontamination pro-
cess by fluorescence microscopy.
1. In order to calculate the amount of microspheres to add to the
drilling mud, it is necessary to determine the volume of the
drilling mud in the tank (see Note 3). Drilling muds are
contained in large mixing tanks, where the liquid is constantly
being mixed to maintain homogeneity and allow for the addi-
tion of drilling mud chemicals as required by the operator.
2. Calculate the volume of fluorescent microspheres needed to
obtain a concentration of 5 105
microspheres/mL in the
drilling mud tank.
3. In order to ensure that the microspheres are dispersed evenly
throughout the drilling mud, first dilute the required volume
of microspheres in a carboy or other similar sterile container
containing deionized water with a sensitivity of 18 MΩ cm at
25
C.

4. Mix well and add to the mud tank while the drilling mud is
being mixed.
5. After mixing, sample drill muds for microsphere quantification
and DNA extraction (see Note 4).
3.1.3 Core Collection Cores from black shale formations contain high amounts of hydro-
carbons and extremely low biomass. In order to obtain samples
adequate for metagenomic analyses, rigorous sterile technique,
proper storage/transport conditions, and minimal time lag before
processing samples are critical.
1. Be on site during the actual collection of core samples. Arrange
with the operator on-site to have access to cores immediately.
2. Label Whirl-Pak bags with pertinent core information, includ-
ing the well, depth, and time of sampling.
3. Photograph each core before inserting in a Whirl-Pak bag
(dimensions dependent on core size), as some cores are recov-
ered intact, while others are recovered in small pieces.
4. If the reservoir is pressurized, it may be necessary to pierce each
Whirl-Pak bag with a thin (25G) needle so that sample bags do
not burst due to de-gassing of the cores.
5. Place Whirl-Pak bags with cores in Anaeropak 7.0 L rectangular
jars, with 3, O2-consuming sachets in each jar.
6. Transport cores in anaerobic boxes on ice, back to the labora-
tory as quickly as possible.
3.2 Contamination
Control in the Lab
Minimize time at room-temp and freeze-thaw cycles. Process sam-
ples as quickly as possible, storing at 4
C during daily processing
steps and store ground samples at 80
C for long-term storage.
3.2.1 Quantification
of Microspheres Prior
to Decontamination
1. Carefully wipe the microscope stage with 70% ethanol.
2. Drill mud samples with microspheres can be quantified by
putting 10–20 μL on a glass slide covered by a glass coverslip.
3. Place the core sample on the microscope stage, being careful to
ensure no drilling mud or core particles contact the objectives.
Microspheres on core samples can be enumerated by taking at
least four images, one on each end of the core, and two images
on each side.
4. Images can be saved using the appropriate software for the
microscope, and counted at a later date, to minimize the time
the sample is exposed to the air.
5. For each image, count the number of microspheres in each field
of view (or multiple, smaller windows from each image).

6. Clean the microscope stage with 70% ethanol between each
sample.
7. Placing autoclaved aluminum foil on the stage with an opening
for the objective helps keep the microscope stage clean and
prevents cross-contamination.
3.2.2 Core Sample
Decontamination
There are many described methods for decontamination of core
samples, including paring via circular saws with hydraulic crushing,
and hammer and/or chisel. Here we determined that the best
results were obtained from three successive NaCl washes while
scrubbing with fine steel wool. Due to the relatively soft nature of
the shale samples from our studies, this resulted in removal of a thin
layer of the exterior, past the point of penetration of microspheres,
and thus microbial contamination.
1. Perform all work in a laminar flow hood dedicated to core
samples.
2. Wipe all surfaces, equipment, and pipettes with 70% ethanol.
3. Put all reagents, supplies, and pipettes in the laminar flow
hood, UV sterilize for 30 min. Rotate pipettes and supplies,
UV sterilize for an additional 30 min.
4. Place three weigh dishes in the laminar flow hood and aliquot
50 mL of 1.5 M NaCl solution in each dish.
5. Place a small piece (2” 2”) of steel wool in each dish.
6. Place the core sample in the first dish, and while wetting the
steel wool, scrub the exterior of the core sample with small
circular motions. You may notice sloughed off core particles in
the NaCl solution.
7. Remove the core from the first wash, and place it in the second
dish. Repeat scrubbing the exterior of the core sample with the
fresh NaCl solution and new steel wool. Repeat the process in
the third wash.
8. Photograph the core sample after the decontamination/clean-
ing process for documentation purposes.
9. Place the core in a new, labeled, Whirl-Pak bag.
of Microspheres Prior
to Decontamination
Repeat the process in Subheading 3.2.1, taking images of the
cleaned core surface. It is likely that no microspheres will be
detected after the decontamination procedure. If microspheres
are observed, repeat the core sample decontamination procedure
in Subheading 3.2.2 and reimage the core until no microspheres are
detected.

3.2.4 Grinding of Core
Samples
samples.
4. Spread a sheet of sterile aluminum foil on the working surface.
5. Light a Bunsen burner near the opening to the laminar flow
hood. Holding the core sample in metal tongs, quickly pass the
core sample through the flame twice while rotating the core
slightly, and place on the aluminum foil sheet in the laminar
flow hood. Allow the core exterior to cool for 1 min.
6. Using a sledge/drilling hammer and cold chisel, break the core
material into smaller pieces, until they will fit in the Plattner’s
mortar sleeve. Place the pestle in the mortar and strike the
pestle with the sledge/drilling hammer. Repeat until the sam-
ple is broken into smaller pieces (see Note 5).
7. Once the sample is broken into small pieces (see Note 6),
transfer the material to the series of stacked sieves, with the
largest mesh at the top. Gently shake with a side-to-side
motion.
8. Remove the material from the base of the stacked sieves and
place in a sterile glass or plastic container for storage.
9. Remove the material in the larger sieves that did not pass
through to the base and place in the Plattner’s mortar and
pestle or a standard ceramic mortar and pestle to further reduce
the size. Pass through the stacked sieves until all sample is
ground to the desired size.
10. Store ground core samples at 80
C until DNA extraction.
3.3 DNA Extraction
and Quantification
from Ground Core
It is essential to use a laminar flow hood dedicated to core samples
during processing. Thoroughly clean the hood before and after any
procedure. The use of extra-long nitrile gloves to cover the exposed
wrist region and disposable Tyvek lab coats is recommended. It is
critical to treat every “blank” exactly as the actual sample and even if
the “blanks” result in non-detectable DNA, to carry the “blanks”
through to library preparation and subsequent sequencing.
Low-level contamination has been well documented even in com-
mercial nucleic acid extraction and purification and amplification
kits [21, 22]. DNA sorption onto mineral surfaces is an additional
problem with low-biomass samples. The use of blocking agents and
carrier molecules have been shown to help overcome this difficulty
[23, 24], and is extensively presented in [16]. However our tests
showed that with these shale core samples, blocking agent and

carrier molecules had deleterious effects on DNA recovery and
additionally contained high levels of microbial contamination.
3.3.1 DNA Extraction
from Ground Core
samples.
4. Turn on the oven to 50
C.
5. Label all tubes, including the same number of tubes for blanks
as for each sample (e.g., if you are extracting from a total 20 g
of ground sample, you will need 40 1.5 mL tubes containing
0.5 g of sample each and 40 1.5 mL tubes for the extraction
blank).
6. Spray all surfaces with (first) DNAzap solution 1, then DNA-
zap solution 2, wipe with sterile paper towels, then with sterile
water.
7. Put 0.5 g ground shale into each 1.5 mL tube (nothing for
blanks). The total number of 0.5 g aliquots is dependent on the
total mass of sample to extract from (see Note 7).
8. Add 1.0 mL of lysis buffer I solution to each tube, vortex to
mix and freeze at 80
C.
9. Thaw samples, vortex, and incubate for 1 h at 50
C, vortexing
every 10 mins.
10. Centrifuge samples at 10k g for 3 min at room temperature,
transfer supernatants from replicate extractions to a 50 mL
conical tube.
11. Put 50 mL tube on ice and extract again from each tube
containing ground shale, and extraction blank tubes, by adding
1.0 mL of lysis buffer I solution, vortex to mix.
12. Centrifuge samples at 10k g for 3 min at room temperature,
transfer supernatants from replicate extractions to a 50 mL
conical tube.
13. Add one volume of phenol:chloroform:isoamyl alcohol to the
combined samples, mix by inversion, centrifuge at 10k g for
10 min at room temperature, transfer supernatant to new
50 mL tube.
14. Add one volume of chloroform:isoamyl alcohol to the samples,
mix by inversion, centrifuge at 10k g for 5 min at room
temperature, transfer supernatant to a new 50 mL tube.
15. Repeat chloroform:isoamyl purification in step 14.

16. Precipitate by adding 30 μL of linear acrylamide and 0.2
volumes of 5 M NaCl solution (see Note 8). Calculate the
new total volume, then add 2.5 volumes of 100% ethanol.
The precipitation may need to be divided between multiple
50 mL conical tubes.
17. Incubate for 2 h at room temperature or overnight at 4
C.
18. Centrifuge samples at 12k g for 30 min at room temperature.
19. Wash 2 with 70% ethanol, centrifuge at max speed for
5–15 min, dry pellet in the laminar flow hood by inverting
tube on sterile aluminum foil.
20. Resuspend pellets in 50–100 μL in warm (50
C) EB buffer.
Use the minimum volume possible to resuspend pellets.
21. Store extracted DNA at 80
C or quantify immediately.
of DNA
It is important to use as small a volume of sample as possible during
quantification. The Qubit dsDNA HS 2.0 fluorometer protocol
allows for up to 20 μL of sample to be quantified. However the
sample is not recoverable after measurement. It is not always neces-
sary to quantify the amount of DNA recovered for extremely
low-biomass samples, as future library preparation for metage-
nomic sequencing can tolerate a wide variety of template concen-
trations. The Qubit dsDNA HS fluorometer 2.0 assay has a
detection limit of 10 pg/μL when using 20 μL of sample. If DNA
concentrations are below the Qubit dsDNA HS detection limit,
10 pg/μL can be used as an upper limit to estimate template input
for library preparation. Other fluorescence-based assays, such as the
Quant-iT PicoGreen dsDNA assay, can also be used but the use of a
nanodrop is not recommended, as some co-extracted contaminants
will cause interference and will inflate the detected concentrations.
2. Put all reagents, supplies, and pipettes (except for samples and
Qubit standards) in the laminar flow hood, UV sterilize for
30 min. Rotate pipettes and supplies, UV sterilize for an addi-
tional 30 min.
3. Thaw DNA on ice.
4. Prepare a master mix according to the manufacture’s instruc-
tions, appropriate for the number of samples to be quantified.
5. Aliquot samples and standards.
6. Measure using the Qubit fluorometer and record detected
DNA concentrations.
7. Store extracted DNA at 80
C or proceed immediately to
metagenomic library preparation.

3.3.3 Library Preparation
and Optional MDA
Amplification
Typical metagenomic library preparations require input DNA con-
centrations ranging from several nanograms up to a microgram. Yet
DNA extractions from samples with low cell density yield DNA
masses in the femtogram to picogram range (1 fg DNA per cell).
Methods have been developed to circumvent low DNA yields, such
as MDA amplification which produces millions of copies of tem-
plate DNA and is used for single-cell genomic amplification; com-
mercial kits such as the Nextera XT DNA library preparation kit
(recommended input 1 ng DNA), and the Accel-NGS 1S Plus
library kit (inputs as low as 10 pg) which was developed for ancient,
degraded and/or ssDNA. Despite the fact that MDA has been
shown to have inherent biases during amplification, it may be
necessary for certain samples when library preparation fails with
non-detectable input DNA; alternatively, the extracted DNA can be
split and libraries prepared with and without MDA amplification.
It is essential to use dedicated laminar flow hoods. Thoroughly
clean the hood before and after any procedure. The use of extra-
long nitrile gloves to cover the exposed wrist region and disposable
Tyvek lab coats is recommended. It is critical to treat every “blank”
exactly as the actual sample and even if the “blanks” result in
non-detectable DNA, to carry the “blanks” through all analyses.
Extreme diligence is required in order to prevent contamination of
samples, and inadvertent amplification of contaminating DNA.
Contamination of a single cell or DNA fragment will distort down-
stream analyses.
1. Carefully read the manufacturer protocols and recommenda-
tions to ensure libraries are representative of the original
sample.
samples.
5. Spray all surfaces with (first) DNAzap solution 1, then DNA-
zap solution 2, wipe with sterile paper towels, then with sterile
water.
6. For optional MDA amplification, follow the manufacturer pro-
tocol with these recommendations:
(a) If performing MDA amplification, be sure to include a
no-template control.
(b) Use the Qiagen “Whole genome amplification from geno-
mic DNA using the REPLI-g Single Cell Kit with
increased sample volumes.” This increases the volume of
template to add from 2.5 to 15 μL.

(c) An incubation time of less than the recommended 8 h may
yield better results. It may be useful to set up reactions
that incubate for 4, 6, and 8 h, and carry all through to
sequencing.
(d) Quantify MDA-amplified DNA using the Qubit protocol
in Subheading 3.3.2. Amplified DNA may need to be
diluted 1:100 or 1:1000 to be in the quantification range.
7. For the Nextera XT DNA library preparation kit, follow the
manufacturer protocol with these recommendations:
(a) Do not overdry the AMPure XP magnetic beads or the
DNA will not be recoverable.
(b) Check the quality and size of library preparation on a
Bioanalyzer High Sensitivity DNA chip.
(c) Contact your sequencing facility to determine if you
should proceed through the Normalize Libraries step.
Many sequencing facilities prefer to perform this step.
4 Notes
1. The drilling process can take days or weeks. It is advantageous
to take samples continuously during the drilling process, espe-
cially when new fluids and chemicals are added to the
drilling muds.
2. Extraction of DNA from drilling muds using the method
described for core samples can result in viscous, unusable
DNA, due to coextraction of drilling mud components. We
obtained the best results using the MoBio PowerMax Soil
DNA isolation kit for drilling muds. Regardless of the DNA
extraction method used, nucleic acids are often still contami-
nated with substances that inhibit PCR reactions or metage-
nomic sequencing due to the complex nature of the samples.
We have found that a secondary cleanup of the nucleic acids is
usually required. The Genomic DNA Clean Concentrator-
10 kit from Zymo Research gives good results, although many
other commercial kits are available. Ethanol precipitation can
have adverse effects, resulting in the concentration of nucleic
acids as well as inhibitory substances.
3. Communication with the operator on-site is critical to obtain
representative samples. Well in advance of sampling, meet with
the operators to determine the amount of site access you’ll
have, and whom to contact during each phase of the operation.
Provide a list of samples needed and instructions for sterile
technique if the operators will take the samples.

4. Bentonite, a clay added to drilling muds in order to protect the
formation from invasion of drilling fluids, has autofluorescent
properties. Be sure to test visualization of microspheres with a
sample of drill mud, before addition. Too much bentonite
coating cores can interfere with visualization of fluorescent
microspheres.
5. Be careful not to strike the Plattner’s pestle when the sleeve is
crooked on the base, or core particles are between the sleeve
and the base. Test by placing the pestle in the sleeve and first
rotating the sleeve on the base. Next rotate the pestle in the
sleeve. If the sleeve and pestle rotate smoothly, the pestle is
seated properly and can be struck with the sledge/drilling
hammer. Failure to properly seat the pestle can result in sample
loss and a bent sleeve, making the Plattner’s pestle unusable.
6. The desired size of core material from the Plattner’s mortar and
pestle depends on the hardness and porosity of each sample
type. For some sample types, it may be easier to first reduce the
size of the core material in the Plattner’s mortar and pestle,
then transfer the material to a standard ceramic mortar and
pestle, using a circular motion to reduce the size further before
sieving. The mesh size of each sieve can be modified based on
the sample type, resulting in larger or smaller particles as
desired.
7. The amount of sample to extract from is dependent on the
sample matrix and the amount of biomass. It may be necessary
to extract from 50–100 g of sample to obtain enough DNA for
sequencing purposes.
8. The final concentration of NaCl during the precipitation pro-
cess is lower than recommended for most protocols, including
the protocol here that we adapted from [16]. We optimized the
concentration of NaCl for our particular samples, as shale con-
tains high amounts of salts due to the fact that they are usually
originally deposited as marine sediments. Tests of precipita-
tions using recommended NaCl concentrations resulted in
recovery of a large salt pellet with little to no quantifiable
DNA recovered. This illustrates the importance of testing all
protocols before performing them on actual samples.
Acknowledgments
Rebecca Daly, Kelly Wrighton and Michael Wilkins were partially
supported by funding from the National Sciences Foundation
Dimensions of Biodiversity (Award No. 1342701) and by the
Marcellus Shale Energy and Environment Laboratory (MSEEL)
funded by Department of Energy’s National Energy Technology
laboratory (DOE-NETL) grant DE#FE0024297.

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stipulate that his name should not appear in the bills—a condition
with which Sir Francis complied, drawing a long breath.
“Mr. O'Brien,” he said, “should the stage ever fail you, a fortune
awaits you if you undertake the duty of teaching gentlemen the art
of being a gentleman.”
“Ah, sir, the moment that art enters the door the gentleman flies
out by the window,” said the actor. “It is Nature, not art, that makes
a gentleman.”
One can well believe that Lady Susan Fox-Strangways, with all the
pride of her connection with a peerage nearly ten years old, treated
Mr. O'Brien's accession to a place in the company of amateurs with
some hauteur, though it was said that she fell in love with him at
once. On consideration, her bearing of hauteur which we have
ventured to assign to her, so far from being incompatible with her
having fallen in love with him, would really be a natural consequence

of such an accident, and the deeper she felt herself falling the more
she would feel it necessary to assert her position, if only for the sake
of convincing herself that it was impossible for her to forget herself
so far as to think of an Irish play-actor as occupying any other
position in regard to her than that of a diversion for the moment.
It was equally a matter of course that Lady Sarah should have an
instinct of what was taking place. She had attended several of the
rehearsals previously in the capacity of adviser to her friend, for
Lady Susan had a high opinion of her critical capacity; but not until
two rehearsals had taken place with O'Brien as Bellaire was she able
to resume her attendance at Downing Street. Before half an hour
had passed this astute lady had seen, first, that O'Brien made every
other man in the cast seem a lout; and, secondly, that Lady Susan
felt that every man in the world was a lout by the side of O'Brien.
She hoped to discover what were the impressions of O'Brien, but
she found herself foiled: the man was too good an actor to betray
himself. The fervour which he threw into the character when making
love to Lady Susan had certainly the semblance of a real passion,
but what did this mean more than that Mr. O'Brien was a convincing
actor?
When she arrived at this point in her consideration of the situation
Lady Sarah lost herself, and began to long with all her heart that the
actor were making love to her—taking her hand with that
incomparable devotion to—was it his art?—which he showed when
Lady Susan's hand was raised, with a passionate glance into her
eyes, to his lips; putting his arm about her waist, while his lips,
trembling under the force of the protestations of undying devotion
which they were uttering, were almost touching Lady Susan's ear.
Before the love scene was over Lady Sarah was in love with the
actor, if not with the man, O'Brien.
So was every lady in the cast. O'Brien was the handsomest actor
of the day. He had been careful of his figure at a time when men of
fashion lived in such a way as made the preservation of a figure
well-nigh impossible. Every movement was grace itself with him, and
the period was one in which the costume of a man gave him every

chance of at least imitating a graceful man. All the others in the cast
of the play seemed imitating the gracefulness of O'Brien, and every
man of them seemed a clown beside him. They gave themselves
countless graces, but he was grace itself.
Lady Sarah saw everything that was to be seen and said nothing.
She was wise. She knew that in due time her friend would tell her all
there was to be told.
She was not disappointed. The play was produced, and of course
every one recognised O'Brien in the part, although the bill—printed
in gold letters on a satin ground, with a charming allegorical design
by Lady Diana Spencer, showing a dozen dainty cupids going to
school with satchels—stated that Bellaire would be represented by “a
gentleman.”
Equally as a matter of course a good many of the spectators
affirmed that it was intolerable that a play-actor should be smuggled
into a company of amateurs, some of them belonging to the best
families. And then to attempt a deception of the audience by
suggesting that O'Brien was a gentleman—oh, the thing was
unheard of! So said some of the ladies, adding that they thought it
rather sad that Lady Susan was not better-looking.
But of the success of the entertainment there could not be a
doubt. It was the talk of the town for a month, and every one
noticed—even her own father—that Lady Susan was looking
extremely thin and very pale.
Lady Sarah said that she had taken the diversion of the theatricals
too seriously.
“I saw it from the first, my dear Sue,” she said.
Sue sprang from her chair, and it would be impossible for any one
to say now that she was over pale.
“You saw it—you—what was it that you saw from the first?” she
cried.
Lady Sarah looked at her and laughed.

“Ah, that is it—what was it that I saw from the first?” she said.
“What I was going to say that I saw was simply that you were
throwing yourself too violently into the production of the play. That
was why you insisted on poor Lord B———'s getting his congé. It
was a mistake—I saw that also.”
“When did you see that?”
“When I saw you taking part in that love scene with Mr. O'Brien.”
“What mean you by that, Lady Sarah?”
“Exactly what you fancy I mean, Lady Susan.”
Lady Susan gazed at her blankly at first, then very pitifully. In
another moment she had flung herself on her knees at the feet of
her friend and was weeping in her lap.
The friend was full of sympathy.
“You poor child!” she murmured, “how could you help it? I vow
that I myself—yes, for some minutes—I was as deep in love with the
fellow as you yourself were. But, of course, you were with him
longer—every day. Lud! what a handsome rascal he is, to be sure.
His lordship must take you to the country without delay. Has the
fellow tried to transfer the character in the play beyond the
footlights?”
“Never—never!” cried Susan. “Sir Francis was right—he is a
gentleman. That is the worst of it!”
“Oh, lud! the worst of it? Are you mad, girl?”
“I am not mad now, but I know that I shall be if he remains a
gentleman—if he refrains from telling me that he loves me—or at
least of giving me a chance of telling him that I love him. That would
be better than nothing—'twould be such a relief. I really do not think
that I want anything more than to be able to confess to him that I
love him—that 'tis impossible that I should love another.”
“The sooner you go to the country the better 'twill be for yourself
and all of us—his lordship especially. Good heavens, child, you must
be mad! Do you fancy that his lordship would give his consent to

your marriage with a strolling player, let him be as handsome as
Beelzebub?”
“He is not a strolling player. Mr. O'Brien is in Mr. Garrick's company,
and every one knows that he is of good family. I have been
searching it out for the past week—all about the O'Briens—there
were a great many of them, all of them distinguished. If it had not
been that King James was defeated by William, in Ireland, Mr.
O'Brien's grandfather would have been made a duke. They were all
heroes, the O'Briens. And they were just too sincere in their
devotion to the losing side—that was it—the losing side was always
the one they took up. And yet you call him a strolling player!”
“I take back the insinuation and offer him my apologies; he is not
a strolling player because he doesn't stroll—would to Heaven he did!
Oh, my poor Sue, take a stroll into the country yourself as soon as
possible and try to forget this dreadfully handsome wretch. You
would not, I am sure, force me to tell his lordship what a goose his
daughter is like to make of herself.”
At this point there was a dramatic scene, one that was far more
deeply charged with comedy of a sort than any to be found in Mr.
Whitehead's play. Lady Susan accused her dear friend of being a spy,
of extorting a confession from her under the guise of friendship,
which in other circumstances—the rack, the wheel, the thumbscrew,
in fact the entire mechanism of persuasion employed by the Spanish
Inquisition—would have been powerless to obtain. Lady Sarah on
her side entreated her friend not to show herself to be even a
greater goose than her confession would make her out to be. For
several minutes there was reproach and counter-reproach, many
home truths followed home thrusts; then some tears, self-
accusation, expressions of sympathy and tenderness, followed by
promises of friendship beyond the dreams of Damon and Pythias;
lastly, a promise on the part of Sue that she would take the advice of
her devoted Sarah and fly to the country without delay.
Strange to say, she fled to the country, and, stranger still, the
result was not to cure her of her infatuation for the handsome actor.
For close upon a year she did not see him, but she was as devoted

to him as she had been at first, and no day passed on which she
failed to think of him, or to spend some hours writing romantic
verses, sometimes in the style of Waller in his lyrics, sometimes in
the style (distant) of Mr. Dryden in his pastorals: she was Lesbia, and
Mr. O'Brien was Strephon.
But in the meantime she had improved so much in her acting that
when Lady Sarah, who had within the year married Sir Thomas
Bunbury, ventured to rally her upon her infatuation of the previous
spring, she was able to disarm her suspicions by a flush and a shrug,
and a little contemptuous exclamation or two.
“Ah, my dear one, did not I give you good advice?” cried Lady
Sarah. “I was well assured that my beloved Sue would never
persevere in a passion that could only end in unhappiness. But
indeed, child, I never had the heart to blame you greatly, the fellow
is handsome as Apollo and as proud as Apolyon. He has broken
many hearts not accounted particularly fragile, during the year.”
“Is't possible? For example?—I vow that I shall keep their names
secret.”
Lady Sarah shook her head at first, but on being importuned
whispered a name or two of ladies of their acquaintance, all of
whom—according to Lady Sarah—had fallen as deep as was possible
in love with O'Brien. Her ladyship was so intent on her narration of
the scandals that she quite failed to see the strange light that
gleamed in her friend's eyes at the mention of every name—a rather
fierce gleam, with a flash of green in it. She did not notice this
phenomenon, nor did she detect the false note in the tribute of
laughter which her friend paid to her powers of narration.
But Lady Sue, when the other had left her, rushed to her room
and flung herself on her bed in a paroxysm of jealousy. She beat her
innocent pillow wildly, crying in the whisper that the clenching of her
teeth made imperative—“The hussies! Shameless creatures! Do they
hope that he will be attracted to them? Fools!—they are fools! They
do not know him as I know him. They think that he is nothing but a
vain actor—Garrick, or Barry, or Lewes. Oh, they do not know him!”

She lay there in her passion for an hour, and if it was her maid
who discovered her at the end of that time, it is safe to assume that
the young woman's flesh was black and blue in places for several
days afterwards. The pinch and the slipper were among the most
highly approved forms of torture inflicted upon their maids at that
robust period of English history. The French Revolution was still
some way off.
A few weeks later Lady Susan was sitting to Sir Joshua Reynolds
for a group, in which he painted her with her friend Lady Sarah
Bunbury and Mr. Henry Fox; and it was the carrying out of this
scheme that put quite another scheme into the quick brain of the
first-named lady. Painting was in the air. She possessed a poor print
of Mr. O'Brien, and she had found an immense consolation in gazing
upon it—frequently at midnight, under the light of her bedroom
candle. The sight of the life-like portraits in Sir Joshua's studio
induced her to ask herself if she might not possess a picture of her
lover that would show him as he really was in life, without
demanding so many allowances as were necessary to be made for
the shortcomings of the engraver of a print. Why should she not get
Sir Joshua Reynolds to paint for her the portrait of Mr. O'Brien?

The thought was a stimulating one, and it took possession of her
for a week. At the end of that time, however, she came to the
conclusion that it would be unwise for her to employ Sir Joshua on a
commission that might possibly excite some comment on the part of
her friends should they come to learn—and the work of this
particular painter was rather inclined to be assertive—that it had
been executed to her order. But she was determined not to live any
longer without a portrait of the man; and, hearing some one
mention at Sir Joshua's house the name of Miss Catherine Read,
who was described as an excellent portrait painter, she made further
inquiry, and the result was that she begged her father, the Earl of

Ilchester, who was devoted to her, to allow her to have her portrait
done by Miss Read, to present to Lady Sarah on her birthday.
Of course Miss Read was delighted to have the patronage of so
great a family—she had not yet done her famous pastel of the
Duchess of Argyll—and Susan, accompanied by her footman, lost no
time in beginning her series of sittings to the artist to whom Horace
Walpole referred as “the painteress.”
She was both patient and discreet, for three whole days elapsed
before she produced a mezzotint of Mr. O'Brien.
“I wonder if you would condescend to draw a miniature portrait of
his lordship's favourite actor from so poor a copy as this, Miss
Read?” she said. “Have you ever seen this Mr. O'Brien—an Irishman,
I believe he is?”
Miss Read assured her that Mr. O'Brien was her favourite actor
also. The print produced was indeed a poor one; it quite failed to do
justice to the striking features of the original, she said.
“I felt certain that it could bear but a meagre resemblance to Mr.
O'Brien if all that I hear of the man be true,” said Lady Susan. “His
lordship swears that there has never been so great an actor in
England, and I should like to give him a surprise by presenting to
him a miniature portrait of his favourite, done by the cunning pencil
of Miss Read, on his birthday. I protest that 'tis a vast kindness you
are doing me in undertaking such a thing. But mind, I would urge of
you to keep the affair a profound secret. I wish it as a surprise to my
father, and its effect would be spoilt were it to become known to any
of his friends that I had this intention.”
“Your ladyship may rest assured that no living creature will hear of
the affair through me,” said the painteress. “But I heartily wish that
your ladyship could procure for me a better copy than this print from
which to work,” she added.
“I fear that I cannot promise you that; I found two other prints of
the same person, but they are worse even than this,” said Lady
Susan. “You must do your best with the material at your disposal.”

“Your ladyship may depend on my doing my best,” replied Miss
Read. “When does his lordship's birthday take place?”
Her ladyship was somewhat taken aback by the sudden question.
It took her some time to recollect that her father's birthday was to
be within a month. She felt that she could not live for longer than
another month without a portrait of the man whom she loved.
While she was going home in her chair she could not but feel that
she had hitherto been an undutiful daughter, never having taken any
interest in her father's birthday, and being quite unacquainted with
its date. She hoped fervently that Miss Read would not put herself to
the trouble to find out exactly on what day of what month it took
place. The result of such an investigation might be a little awkward.
It so happened that Miss Read took no trouble in this direction. All
her attention was turned upon the task of making a presentable
miniature out of the indifferent material with which she had been
supplied for this purpose. She began wondering if it might not be
possible to get O'Brien to sit to her half a dozen times in order to
give her a chance of doing credit to herself and to the gentleman's
fine features.
She was still pondering over this question when her attendant
entered with a card, saying that a gentleman had come to wait on
her.
She read the name on the card, and uttered an exclamation of
surprise, for the name was that of the man of whom she was
thinking—Mr. O'Brien, of Drury Lane Theatre.
She had wholly failed to recover herself before he entered the
studio, and advanced to her, making his most respectful bow. He
politely ignored her flutter-ings—he was used to see her sex
overwhelmed when he appeared.
“Madam, I beg that you will pardon this intrusion,” he said. “I have
taken the liberty of waiting upon you, knowing of your great capacity
as an artist.”
“Oh, sir!” cried the fluttered little lady, making her courtesy.

“Nay, madam, I have no intention of flattering one to whom
compliments must be as customary as they are well deserved,” said
the actor. “I come not to confer a favour, madam, but to entreat
one. In short, Miss Read, I am desirous of presenting a valued friend
of mine with the portrait of a lady for whom he entertains a sincere
devotion. For certain reasons, which I need not specify, the lady
cannot sit to you; but I have here a picture of her poorly done in
chalks, from which I hope it may be in your power to make a good—
a good—— Good heavens! what do I behold? 'Tis she—she—Lady
Susan herself!”
He had glanced round the studio in the course of his speech, and
his eyes had alighted upon the newly-begun portrait of Lady Susan.
It represented only a few days' work, but the likeness to the original
had been ably caught, and no one could fail to recognise the
features.
He took a hurried step to the easel, and the air made by his
motion dislodged a print which the artist had laid on the little ledge
that supported the stretcher of the canvas. The print fluttered to the
floor; he picked it up, and gave another exclamation on recognising
his own portrait in the mezzotint.
Looking from the print to the picture and then at Miss Read, he
said in a low voice, after a pause—“Madam, I am bewildered. Unless
you come to my assistance I protest I shall feel that I am dreaming
and asleep. Pray, madam, enlighten me—for Heaven's sake tell me
how this”—he held up the print—“came into such close juxtaposition
with that”—he pointed to the portrait on the easel.
“'Tis easily told, sir,” said Miss Read, smiling archly. “But I must
leave it to your sense of honour to keep the matter a profound
secret.”
“Madam,” said Mr. O'Brien with dignity, “Madam, I am an
Irishman.”
“That is enough, sir; I know that I can trust you. The truth is, Mr.
O'Brien, that Lady Susan is sitting to me for her portrait—that

portrait. 'Twas marvellous that you should recognise it so soon. I
have not worked at it for many hours.”
“Madam, your art is beyond that of the magician. 'Tis well known
that every form depicted by Miss Read not only breathes but
speaks.”
“Oh, sir, I vow that you are a flatterer; still, you did recognise the
portrait—'tis to be presented to Lady Sarah Bunbury.”
“Her ladyship will be the most fortunate of womankind.”
“Which ladyship, sir—Lady Susan or Lady Sarah?”
“Both, madam.” The Irishman was bowing with his hand on his
heart. “But the print—my poor likeness?”
“That is the secret, sir; but you will not betray it when I tell you
that Lady Susan entrusted that print to me in order that I might
make a copy in miniature for her to present to her father, Lord
Ilchester. You are his favourite actor, Mr. O'Brien, as no doubt you
are aware.”
“'Tis the first I heard of it, madam.” There was a suggestion of
mortification in the actor's tone.
“Ah, 'twould be impossible for Mr. O'Brien to keep an account of all
his conquests. But now you can understand how it is that her
ladyship wishes her intention to be kept a secret: she means to add
to the acceptability of her gift by presenting it as a surprise. But her
secret is safe in your keeping, sir?”
“I swear to it, madam.” Mr. O'Brien spoke mechanically. His hand
was on his chin: he was clearly musing upon some question that
perplexed him. He took a turn up and down the studio, and then
said:
“Madam, it has just occurred to me that you, as a great artist——”
“Nay, sir,” interposed the blushing painteress.
“I will not take back a word, madam,” said the actor, holding up
one inexorable hand. “I say that surely so great an artist as you
should disdain to do the work of a mere copyist. Why should not you

confer upon me the honour of sitting to you for the miniature
portrait?”
“Oh, sir, that is the one favour which I meant to ask of you, if my
courage had not failed me.”
“Madam, you will confer immortality upon a simple man through
that magic wand which you wield.” He swept his hand with inimitable
grace over the mahl-stick which lay against the easel. “I am all
impatient to begin my sitting, Miss Read. Pray let me come to-
morrow.”
“Her ladyship comes to-morrow.”
“I shall precede her ladyship. Name the hour, madam.”
Without the least demur Miss Read named an hour which could
enable him to be far away from the studio before Lady Susan's
arrival.
And yet the next day Lady Susan entered the studio quite half an
hour before Mr. O'Brien had left it. Of course she was surprised. Had
not Miss Read received a letter, making her aware of the fact that
she, Lady Susan, would be forced, owing to circumstances over
which she had no control, to sit for her portrait an hour earlier than
that of her appointment?
When Miss Read said she had received no such letter, Lady Susan
said some very severe things about her maid. Miss Read was greatly
fluttered, but she explained in as few words as possible how it was
that Mr. O'Brien had come forward in the cause of art, and was
sitting for the miniature. Lady Susan quickly got over her surprise.
(Had Miss Read seen the letter which her ladyship had received the
previous evening from Mr. O'Brien she would not have marvelled as
she did at the rapidity with which her ladyship recovered her self-
possession.) Her ladyship was quite friendly with the actor, and
thanked him for his courtesy in offering to give up so much of his
time solely for the sake of increasing the value of her gift to her
father.
A few minutes later, while they were discussing some point in the
design of the picture, Miss Read was called out of the studio, and in

a second Lady Susan was in his arms.
“Fate is on our side, darling girl!” he whispered.
“I could not live without you, my charmer. But I was bold! I took
my fate in both hands when I wrote you that letter.”
“Dear one, 'twas the instinct of true love that made you guess the
truth—that I wanted the portrait because I loved the original. Oh,
dear one, what have I not suffered during the year that has parted
us!” said Lady Susan, with her head upon his shoulder.
The Irishman found it necessary to fall back upon the seductive
tongue of his country for words of endearment to bestow upon her.
He called her “Sheila,” “a cushla machree,” “mavourneen,” and also
“aroon.” But when Miss Read returned to the studio they were still
discussing a purely artistic point in connection with the portrait.
Of course now that O'Brien knew the secret of the miniature there
was no reason that Miss Read could see why he and Lady Susan
should not meet at her studio. To do her justice, neither could her
ladyship perceive why they should not come together at this place.
They came every day, and every day Lady Susan begged that Miss
Read would allow her to rest in her ante-room after the fatigue of
the sitting. She rested in that room, and in the company of O'Brien,
until at last Miss Read became frightened; and one day told her
friend Lord Cathcart something of her fears. Lord Cathcart, in his
turn, told Lord Ilchester. His lordship was furious, but cautious.
He wanted evidence of his daughter's infatuation. He got it the
next morning, for he insisted on seeing a letter which arrived for
Lady Susan, addressed in the handwriting of Lady Sarah. This letter
turned out to be from O'Brien, and Susan confessed that her father's
surmise was correct—all the letters which she had recently received
in Lady Sarah's hand had come from O'Brien.
Her father was foolish enough to grant her permission to say
farewell to her lover, and thus the two were allowed to come
together once more. They had a long talk, in the course of which
O'Brien communicated to her a secret of the theatre, which was that
Mr. Garrick and Mr. Colman were engaged in the construction of a

comedy to be called The Clandestine Marriage, and that Mr. Garrick
told him that he, O'Brien, was to play the part of the lover—the
gentleman who had married the lady in secret.
Lady Susan parted from her lover, not in tears, but in laughter.
The conclusion of the story is told by Horace Walpole, writing to
Lord Hertford.
“You will have heard of the sad misfortune that has happened to
Lord Ilchester by his daughter's marriage with O'Brien, the actor,”
wrote Walpole; and then went on to tell how Lady Susan had made
her confession to her father, vowing to have nothing more to do with
her lover if she were but permitted to bid him good-bye. “You will be
amazed,” continued Walpole, “even this was granted. The parting
scene happened the beginning of the week. On Friday she came of
age, and on Saturday morning—instead of being under lock and key
in the country—walked downstairs, took her footman, said she was
going to breakfast with Lady Sarah, but would call at Miss Read's; in
the street pretended to recollect a particular cap in which she was to
be drawn, sent the footman back for it, whipped into a hackney
chair, was married at Covent Garden Church, and set out for Mr.
O'Brien's villa at Dunstable.”
Unlike many other alliances of a similar type, this marriage turned
out a happy one. O'Brien was induced to leave the stage and to
depart with his wife for America. He obtained a grant of some forty
thousand acres in the province of New York, and had he retained
this property and taken the right side during the Revolution his
descendants would to-day be the richest people in the world. A few
years later he was given a good appointment in Bermuda; and
finally, in 1770, he was made Receiver-General of the County of
Dorset, and became popular as a country squire. He died in 1815,
and Lady Susan survived him by twelve years.
It was Lady Sarah who had made the imprudent marriage. She
submitted to the cruelties of her husband for fourteen years, and on
her leaving his roof he obtained a divorce.

In 1781, nineteen years after her first marriage, she wedded the
Hon. George Napier, and became the mother of three of the greatest
Englishmen of the nineteenth century. She lived until she was eighty.
Her friend Lady Susan followed her to the grave a year later, at the
age of eighty-four.
THE END

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