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DNA MICROARRAYS
AND RELATED
GENOMICS
TECHNIQUES
Design, Analysis,
and Interpretation
of Experiments
 Biostatistics: A Series of References and Textbooks

Series Editor
Shein-Chung Chow
Duke Clinical Research Institute
Duke University
Durham, NC, USA

1. Design and Analysis of Animal Studies in Pharmaceutical Development,

Shein-Chung Chow and Jen-pei Liu
2. Basic Statistics and Pharmaceutical Statistical Applications,
James E. De Muth
3. Design and Analysis of Bioavailability and Bioequivalence Studies,
Second Edition, Revised and Expanded, Shein-Chung Chow and Jen-pei Liu
4. Meta-Analysis in Medicine and Health Policy, Dalene K. Stangl and
Donald A. Berry
5. Generalized Linear Models: A Bayesian Perspective, Dipak K. Dey,
Sujit K. Ghosh, and Bani K. Mallick
6. Difference Equations with Public Health Applications, Lemuel A. Moyé
and Asha Seth Kapadia
7. Medical Biostatistics, Abhaya Indrayan and Sanjeev B. Sarmukaddam
8. Statistical Methods for Clinical Trials, Mark X. Norleans
9. Causal Analysis in Biomedicine and Epidemiology: Based on Minimal
Sufficient Causation, Mikel Aickin
10. Statistics in Drug Research: Methodologies and Recent Developments,
Shein-Chung Chow and Jun Shao
11. Sample Size Calculations in Clinical Research, Shein-Chung Chow, Jun Shao,
and Hansheng Wang
12. Applied Statistical Design for the Researcher, Daryl S. Paulson
13. Advances in Clinical Trial Biostatistics, Nancy L. Geller
14. Statistics in the Pharmaceutical Industry, 3rd Edition, Ralph Buncher
and Jia-Yeong Tsay
15. DNA Microarrays and Related Genomics Techniques: Design, Analysis, and
Interpretation of Experiments, David B. Allsion, Grier P. Page, T. Mark
Beasley, and Jode W. Edwards
DNA MICROARRAYS
AND RELATED
GENOMICS
TECHNIQUES
Design, Analysis,
and Interpretation
of Experiments

EDITED BY
DAVID B. ALLISON
GRIER P. PAGE
T. MARK BEASLEY
JODE W. EDWARDS

Boca Raton London New York

Published in 2006 by
Chapman & Hall/CRC
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

© 2006 by Taylor & Francis Group, LLC

Chapman & Hall/CRC is an imprint of Taylor & Francis Group
No claim to original U.S. Government works
Printed in the United States of America on acid-free paper
10 9 8 7 6 5 4 3 2 1
International Standard Book Number-10: 0-8247-5461-1 (Hardcover)
International Standard Book Number-13: 978-0-8247-5461-7 (Hardcover)
Library of Congress Card Number 2005050488
This book contains information obtained from authentic and highly regarded sources. Reprinted material is
quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts
have been made to publish reliable data and information, but the author and the publisher cannot assume
responsibility for the validity of all materials or for the consequences of their use.
No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and
recording, or in any information storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copyright.com
(http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive,
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system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only
for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data

DNA microarrays and related genomics techniques : design, analysis, and interpretation of experiments
/ editors, David B. Allison ... [et al.].
p. cm. -- (Biostatistics)
Includes bibliographical references and index.
ISBN 0-8247-5461-1 (alk. paper)
1. DNA microarrays. I. Allison, David B. (David Bradley), 1963- II. Biostatistics (New York, N.Y.)

QP624.5.D726.D636 2005
572.8'636--dc22 2005050488

Visit the Taylor & Francis Web site at

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Taylor & Francis Group and the CRC Press Web site at
is the Academic Division of Informa plc. http://www.crcpress.com
Series Introduction
The primary objectives of the Biostatistics Book Series are to provide useful ref-
erence books for researchers and scientists in academia, industry, and government,
and also to offer textbooks for undergraduate and graduate courses in the area of
biostatistics and bioinformatics. This book series will provide comprehensive and
unified presentations of statistical designs and analyses of important applications in
biostatistics and bioinformatics, such as those in biological and biomedical research.
It gives a well-balanced summary of current and recently developed statistical meth-
ods and interpretations for both statisticians and researchers/scientists with minimal
statistical knowledge who are engaged in the field of applied biostatistics and bioin-
formatics. The series is committed to providing easy-to-understand, state-of-the-art
references and textbooks. In each volume, statistical concepts and methodologies will
be illustrated through real world examples whenever possible.
In recent years, the screening of thousands of genes using the technique of
expression microarrays has become a very popular topic in biological and biomedical
research. The purpose is to identify those genes that may have an impact on clinical
outcomes of a subject who receives a test treatment under investigation and con-
sequently establish a medical predictive model. Under a well-established predictive
model, we will be able not only to identify subjects with certain genes who are most
likely to respond to the test treatment, but also to identify subjects with certain genes
who are most likely to experience (serious) adverse events. This concept plays an
important role in the so-called personalized medicine research. This volume summar-
izes various useful experimental designs and statistical methods that are commonly
employed in microarray studies. It covers important topics in DNA microarrays and
related genomics research such as normalization of microarray data, microarray qual-
ity control, statistical methods for screening of high-dimensional biology, and power
and sample size calculation. In addition, this volume provides useful approaches to
microarray studies such as clustering approaches to gene microarray data, paramet-
ric linear models, nonparametric procedures, and Bayesian analysis of microarray
data. It would be beneficial to biostatisticians, biological and biomedical researchers,
and pharmaceutical scientists who are engaged in the areas of DNA microarrays and
related genomics research.

Shein-Chung Chow
Preface
WHAT ARE MICROARRAYS?
Microarrays have become a central tool used in modern biological and biomedical
research. This book concerns expression microarrays, which for the remainder of the
book, we simply refer to as microarrays. They are tools that permit quantification of
the amount of all mRNA transcripts within a particular biological specimen. There are
several different technologies for producing microarrays that have different strengths
and weaknesses. These platforms and alternatives are discussed in Chapter 1 by
Gaffney et al.
Viewed as “hot” and highly exotic tools as recently as the late 1990s, they are now
ubiquitous in biological research and the modern biological researcher can no more
be unaware and unexposed to microarray research and its results than one can remain
ignorant of clinical trials, questionnaire studies, genome scans, animal models, or any
of the other tools that have become standard parts of our armamentarium. Although
much development in microarray research methodology is still needed, it is clear that
microarrays are here to stay.

WHY THIS BOOK?

In one sense, microarrays are simply measurement assays. Just as one can meas-
ure, for example, the amount of insulin (which is the product of a gene) in blood,
we can measure the products of genes with microarrays in any tissue. What distin-
guishes microarrays from traditional approaches is their “omic” nature. That is, they
have capacity to measure all gene transcripts at once. This ushered in the subfield
of transcriptomics. A particular challenge is that because of the expense of micro-
array research and the fact that it is often directed at basic discovery and hypothesis
generation/exploration missions, the number of variables (transcripts) available in
microarray studies tends to exceed the number of cases (subjects) by several orders
of magnitude. Traditional statistical approaches to design and analysis were not
developed in the context of such high dimensional and small sample problems. We
and many others now find that our training in traditional statistical methods is not
especially well-suited to such situations.
We (the editors) were first introduced to the analysis of microarray data ca.
1999. At that time, there were almost no statistical papers providing approaches
to analyze microarray data or design microarray studies from a statistical perspect-
ive. By 2003, this situation had changed dramatically and we estimate that there
were hundreds of papers thereon (Mehta et al., 2004). This overwhelming deluge
of methods from these papers is quite daunting to either the applied investigator
looking for methodologies to utilize or the methodologist trying to keep up with the
field.
 As part of the research efforts funded by the National Science Foundation, we have
hosted an annual retreat for scientists interested in analytic methods for microarray
research for the last five years. The impetus for this book came in part from discussions
held at those retreats. We felt there was a need for a book that consolidated many of
the existing methodologic advances and compiled many of the issues and methods
into a single volume. This book is aimed at both the investigator who will conduct
analyses of microarray data and at the methodologists who will evaluate existing and
develop future methodologies.

WHAT IS HERE?
We have structured this book in a manner that we believe parallels the steps that an
investigator or an analyst will go through while conducting and analyzing a microarray
experiment from conception to interpretation. We begin with the most foundational
issues: ensuring the quality and integrity of the data and assessing the validity of the
statistical methods we employ. We then move on to the often neglected, but critical
aspects of designing a microarray experiment. Gadbury et al. (Chapter 5) address
issues such as power and sample size, where only very recently have developments
allowed such calculations in a high dimensional context. The third section of the book
is the largest, addressing issues of the analysis of microarray data. The size of this
section reflects both the variety of topics and the amount of effort investigators have
devoted to developing new methodologies. Finally, we move on to the intellectual
frontier — interpretation of microarray data. New methods for facilitating and affect-
ing formalization of the interpretation process are discussed. The movement to make
large high dimensional datasets public for further analysis and methods for doing so
are also addressed.

WHAT IS NOT HERE?

This book is not a detailed exposition of software packages (although some are men-
tioned in specific chapters), biochemistry, or the mechanics of the physical production
of microarrays or biological specimens for analysis via microarrays. Interested read-
ers should consult other more topical books in these areas (Jordan, 2001; Grigorenko,
2002; Ye and Day, 2003) Many closely related disciplines such as proteomics and
metabolomics are not discussed in any depth although the astute reader will readily
see the commonalities among the statistical and design approaches that can be applied
to such data.

THE FUTURE
There is no question that this field will continue to advance rapidly and some of the
specific methodologies we discuss herein will be replaced by new advances in the
near future. Nevertheless, we believe the field is now at a point where a foundation
of key categories of methods has been laid and begun to settle. Although the details
may change, we believe that the majority of the key principles described herein and
the foundational categories are likely to stand the test of time and serve as a useful
guide to the reader. We look forward to new biological knowledge that we anticipate
will emerge from the evermore sophisticated technologies and analysis as well as the
exciting new statistical advances sure to come.

REFERENCES
Girgorenko E.V. (2002) DNA Arrays: Technologies and Experimental Strategies.
CRC Press, Boca Raton, FL.
Jordan B.R. (ed.) (2001) DNA Microarray: Gene Expression Applications. Springer-Verlag,
Berlin.
Mehta T., Tanik M., and Allison D.B. (2004) Towards sound epistemological foundation of
statistical methods for high-dimensional biology. Nature Genetics 36: 943–947.
Ye S. and Day I.N.M. (2003) Microarray and Microplates. Bios Press, Oxford.
Editors
David B. Allison received his Ph.D. from Hofstra University in 1990. He then
completed a postdoctoral fellowship at the Johns Hopkins University School of Medi-
cine and a second postdoctoral fellowship at the NIH-funded New York Obesity
Research Center at St. Luke’s/Roosevelt Hospital Center. He was a research scient-
ist at the New York Obesity Research Center and Associate Professor of Medical
Psychology at Columbia University College of Physicians and Surgeons until 2001.
In 2001, he joined the faculty of the University of Alabama at Birmingham where
he is currently Professor of Biostatistics, Head of the Section on Statistical Genet-
ics, and Director of the NIH-funded Clinical Nutrition Research Center. He has
authored over 300 scientific publications and edited three books. He has won sev-
eral awards, including the 2002 Lilly Scientific Achievement Award from the North
American Association for the Study of Obesity and the 2002 Andre Mayer Award
from the International Association for the Study of Obesity, holds several NIH and
NSF grants, served on the Council of the North American Association for the Study
of Obesity from 1995 to 2001, and has been a member of the Board of Trustees
for the International Life Science Institute, North America, since January 2002. He
serves on the editorial boards of Obesity Reviews; Nutrition Today; Public Library
of Science (PLOS) Genetics; International Journal of Obesity; Behavior Genetics;
Computational Statistics and Data Analysis; and Human Heredity.
Dr. Allison’s research interests include obesity, quantitative genetics, clinical
trials, and statistical and research methodology.

Grier P. Page, Ph.D. was born in Cleveland, Ohio in 1970. He received his B.S.
in Zoology and Molecular Biology from the University of Texas, Austin. Then he
received his M.S. and Ph.D. in Biomedical Sciences from the University of Texas–
Health Sciences Center—Houston under the mentorship of Drs. Eric Boerwinkle and
Christopher Amos. Dr. Page has been involved in the use and analysis of microarrays
since 1998 for expression, genomics, and genotyping. He is very active in the devel-
opment of new methods for the analysis of microarray data as well as methods and
techniques for the generation on the highest quality microarray data. He uses microar-
rays in his research in the mechanisms of cancer development, nutrient production,
and nutrient gene interactions especially in cancer and plants. He is currently a mem-
ber of the Section on Statistical Genetics, Department of Biostatistics the University
of Alabama, Birmingham.

T. Mark Beasley, Ph.D. is Associate Professor of Biostatics and a member of the

Section on Statistical Genetics at the University of Alabama at Birmingham. He is
the leader of the measurement and inferences teams for a funded National Science
Foundation (NSF) grant to further the development of microarray analysis methods.
He has a Ph.D. in Statistics and Measurement from Southern Illinois University and
a strong research record in the area of statistical methodology, focused in methodo-
logical problems in statistical genetics; nonparametric statistics; simulation studies;
and the use of linear models. He also has a strong background in measurement the-
ory and the multivariate methods (e.g., factor analysis, structural equation models;
regression models). Dr. Beasley teaches courses on Applied Multivariate Analysis and
General Linear Models at UAB and is currently Editor of Multiple Linear Regression
Viewpoints, a journal focused on applications of general linear models and mul-
tivariate analysis. He has published articles in applied statistics journals such as the
Journal of Educational & Behavioral Statistics, Journal of the Royal Statistical Soci-
ety, Computational Statistics & Data Analysis, Multivariate Behavioral Research,
and Communications in Statistics. He has also published articles on methodological
problems in statistical genetics in leading journals such as the American Journal of
Human Genetics; Behavior Genetics; Genetic Epidemiology; Genetics, Selection,
and Evolution and Human Heredity.

Jode W. Edwards received a Ph.D. in plant breeding and genetics with a minor in
statistics from Iowa State University in 1999. He then spent 3 years with Monsanto
Company as a statistical geneticist working in the areas of marker-assisted plant
breeding and QTL mapping. Dr. Edwards joined the Section on Statistical Genetics as
a Postdoctoral Fellow in 2002. His research involved application of Empirical Bayes
methods to microarray analysis and development of software for microarray data
analysis. Using SAS as a prototyping platform, he designed experimental versions of
the HDBStat! software that is now distributed by the Section on Statistical Genetics.
Additionally, Dr. Edwards helped initiate efforts to build the microarray Power Atlas, a
tool to assist investigators in designing microarray experiments. In 2004, he completed
his postdoctoral studies and assumed a position as a Research Geneticist with the
Agricultural Research Service of the United States Department of Agriculture, in
Ames, IA. His research is focused on quantitative genetics of maize, application of
Bayesian methods in plant breeding, and breeding for amino acid balance in maize
protein.
Contributors

David B. Allison Robert R. Delongchamp

Department of Biostatistics Division of Biometry and Risk
University of Alabama at Birmingham Management
Birmingham, Alabama National Center for Toxicological
Research
T. Mark Beasley Jefferson, Arizona
Department of Biostatistics
Shibing Deng
University of Alabama at Birmingham
SAS Institute
Birmingham, Alabama
Cary, North Carolina

Jacob P.L. Brand Jode W. Edwards

Department of Biostatistics Department of Biostatistics
University of Alabama at Birmingham University of Alabama at Birmingham
Birmingham, Alabama Birmingham, Alabama

Jane Y. Chang David Finkelstein

Department of Applied Statistics and Hartwell Center
Operational Research St. Jude Children’s Research Hospital
Bowling Green State University Memphis, Tennessee
Bowling Green, Ohio
Gary L. Gadbury
Department of Mathematics and Statistics
Kei-Hoi Cheung University of Missouri-Rolla
Department of Genetics Rolla, Missouri
Center for Medical Informatics
Yale University School of Medicine Patrick M. Gaffney
New Haven, Connecticut University of Minnesota
Minneapolis, Minnesota
Tzu-Ming Chu
SAS Institute Elizabeth Garrett-Mayer
Cary, North Carolina Division of Oncology Biostatistics
Sidney Kimmel Comprehensive
Cancer Center
Christopher S. Coffey
Baltimore, Maryland
University of Alabama at Birmingham
Birmingham, Alabama Pulak Ghosh
Department of Mathematics and
Stacey S. Cofield Statistics
University of Alabama at Birmingham Georgia State University
Birmingham, Alabama Atlanta, Georgia
Bernard S. Gorman Michael V. Osier
Nassau Community College and Yale Center for Medical Informatics
Hofstia University Yale University School of Medicine
Garden City, New York New Haven, Connecticut

Jason C. Hsu Grier P. Page

Department of Statistics Department of Biostatistics
Ohio State University University of Alabama at Birmingham
Columbus, Ohio Birmingham, Alabama

Michael Janis Rudolph S. Parrish

Department of Chemistry, Department of Bioinformatics and
Biochemistry, and Biostatistics School of Public
Molecular Biology Health and Information Sciences
University of California University of Louisville
at Los Angeles Louisville, Kentucky
Los Angeles, California
Jacques Retief
Christina M. Kendziorski Iconix Pharmaceuticals
Department of Biostatistics and Mountain View, California
Medical Informatics
University of Wisconsin-Madison
Douglas M. Ruden
Madison, Wisconsin
Department of Environmental Health
Sciences
Jeanne Kowalski
University of Alabama at Birmingham
Division of Oncology Biostatistics
Birmingham, Alabama
Johns Hopkins University
Baltimore, Maryland
Chiara Sabatti
Jeffrey D. Long Department of Human Genetics
Department of Biostatistics University of California at Los Angeles
University of Alabama at Birmingham Los Angeles, California
Birmingham, Alabama
Kathryn Steiger
Tapan Mehta Division of Biostatistics
Department of Biostatistics University of California at Berkeley
University of Alabama at Birmingham Berkeley, California
Birmingham, Alabama
Murat Tanik
Kathy L. Moser Department of Biostatistics
Department of Medicine University of Alabama at Birmingham
Institute of Human Genetics and Birmingham, Alabama
Center for Immunology
University of Minnesota Alan Williams
Medical School Affymetrix
Minneapolis, Minnesota Santa Clara, California
Russell D. Wolfinger Kui Zhang
SAS Institute Department of Biostatistics
Cary, North Carolina University of Alabama at Birmingham
Birmingham, Alabama
Qinfang Xiang
Department of Mathematics and Zhen Zhang
Statistics Department of Pathology
University of Missouri-Rolla School of Medicine
Rolla, Missouri Johns Hopkins University
Stanislav O. Zakharkin Baltimore, Maryland
Department of Biostatistics
University of Alabama at Birmingham
Birmingham, Alabama
Contents
Chapter 1
Microarray Platforms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Patrick M. Gaffney and Kathy L. Moser

Chapter 2
Normalization of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Rudolph S. Parrish and Robert R. Delongchamp

Chapter 3
Microarray Quality Control and Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
David Finkelstein, Michael Janis, Alan Williams, Kathryn Steiger, and
Jacques Retief

Chapter 4
Epistemological Foundations of Statistical Methods for High-Dimensional
Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Stanislav O. Zakharkin, Tapan Mehta, Murat Tanik, and David B. Allison

Chapter 5
The Role of Sample Size on Measures of Uncertainty and Power . . . . . . . . . . . . . . . . 77
Gary L. Gadbury, Qinfang Xiang, Jode W. Edwards, Grier P. Page, and
David B. Allison

Chapter 6
Pooling Biological Samples in Microarray Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Christina M. Kendziorski

Chapter 7
Designing Microarrays for the Analysis of Gene Expressions . . . . . . . . . . . . . . . . . . . . 111
Jane Y. Chang and Jason C. Hsu

Chapter 8
Overview of Standard Clustering Approaches for Gene Microarray Data
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Elizabeth Garrett-Mayer

Chapter 9
Cluster Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Bernard S. Gorman and Kui Zhang
Chapter 10
Dimensionality Reduction and Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Jeanne Kowalski and Zhen Zhang

Chapter 11
Modeling Affymetrix Data at the Probe Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Tzu-Ming Chu, Shibing Deng, and Russell D. Wolfinger

Chapter 12
Parametric Linear Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Christopher S. Coffey and Stacey S. Cofield

Chapter 13
The Use of Nonparametric Procedures in the Statistical Analysis of
Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
T. Mark Beasley, Jacob P.L. Brand, and Jeffrey D. Long

Chapter 14
Bayesian Analysis of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Jode W. Edwards and Pulak Ghosh

Chapter 15
False Discovery Rate and Multiple Comparison Procedures . . . . . . . . . . . . . . . . . . . . . . 289
Chiara Sabatti

Chapter 16
Using Standards to Facilitate Interoperation of Heterogeneous Microarray
Databases and Analytic Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Kei-Hoi Cheung

Chapter 17
Postanalysis Interpretation: “What Do I Do with This Gene List?” . . . . . . . . . . . . . . 321
Michael V. Osier

Chapter 18
Combining High Dimensional Biological Data to Study Complex Diseases
and Quantitative Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Grier P. Page and Douglas M. Ruden

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
 1 Microarray Platforms

Patrick M. Gaffney and Kathy L. Moser

CONTENTS

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Microarray Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.3 Autoantigen and Cytokine Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.4 DNA and Oligonucleotide Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Tiling Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.6 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.7 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.1 INTRODUCTION
As with the development of any novel and potentially powerful technology, the
prospect of revealing new information that may dramatically change our under-
standing of biological processes can generate much excitement. Such is true for the
emerging genomic approaches that make possible high-density assays using micro-
array platforms. Indeed, it is difficult, if not impossible, to imagine any area of biology
that could not be affected by the wide range of potential applications of microarray
technology. Numerous examples, such as those from the field of oncology, provide
striking evidence of the power of microarrays to bring about extraordinary advances in
molecularly defining important disease phenotypes that were otherwise unrecognized
using conventional approaches such as histology.
In this chapter, we present a general overview of microarray platforms currently
in use with particular emphasis on high-density DNA arrays. We touch briefly on
approaches to data analysis leaving most of the details for the ensuing chapters. For
those just entering the microarray arena or interested in more details, a series of
particularly useful reviews have recently been published that take stock of the latest
developments and discuss the most pressing challenges of this technology [1].

1.2 MICROARRAY TECHNOLOGY

Microarray technology provides an unprecedented and uniquely comprehensive probe
into the coordinated workings of entire biological pathways and genomic-level

1
2 DNA Microarrays and Related Genomics Techniques

TABLE 1.1
Potential Objectives of Studies
Utilizing Microarray Technology
1. Distinguish patients from normal controls
2. Identify subsets of patients
3. Characterize host responses
4. Examine cellular pathways
5. Compare alternative experimental conditions
6. Examine drug response
7. Follow temporal changes in gene expression
8. Identify candidate genes for genetic studies

processes. In general terms, microarrays refer to a variety of platforms in which

high density assays are performed in parallel on a solid support. Thousands to tens
of thousands of datapoints may be generated in each experiment. The growth of
scientific literature since the mid-1990s may provide some indication for the poten-
tial impact of this technology in biomedical sciences. A majority of applications have
been in oncology, although many examples from other fields are rapidly emerging and
include examination of host response to pathogens, examination of drug responses,
identification of temporal changes in gene expression, and comparisons of various
experimental conditions.
Three major types of microarrays exist — tissue, protein, and DNA. Tissue
microarrays immobilize small amounts of tissue from biopsies of multiple subjects
on glass slides for immunohistochemical processing, while protein arrays immobilize
peptides or intact proteins for detection by antibodies or other means (see Section 1.3).
For the last several years, much excitement and attention has focused on DNA micro-
arrays and most of this book will concentrate on DNA microarray analysis. Regardless
of the specific platform used, these approaches offer new opportunities to address bio-
logic questions in a way never possible before. Table 1.1 provides just a few examples
of the potential ways in which microarray technology can be utilized.

1.3 AUTOANTIGEN AND CYTOKINE MICROARRAYS

Applications of protein microarrays include assessment of enzyme–substrate,
protein–protein, and DNA–protein interactions. Although efforts to develop these pro-
teomic tools predate the first descriptions of DNA microarrays [2], progress has been
relatively slower — in part due to challenges posed by natural inherent differences
in proteins compared with DNA. As examples, proteins consist of highly diverse
conformational structures that result from 20 amino acids vs. the 4 nucleic acid build-
ing blocks that generate a relatively uniform structure in DNA. Proteins may exist
as large complexes, can be hydrophilic or hydrophobic, acidic or basic, and contain
Microarray Platforms 3

posttranslational modifications such as acetylation, glycosylation, or phosphoryla-

tion. Functional and conformational properties of proteins must often remain intact
when immobilized onto a microarray in order to retain the desired binding properties
for detection of target ligands.
The development of protein microarrays to detect immunologic targets such
as cytokines or autoantibodies has enormous potential for research and diagnostic
applications in autoimmune diseases. Several groups, including Joos and col-
leagues in Germany [3], and Robinson and colleagues at Stanford University [4],
have made important strides in developing autoantigen microarrays for multiplex
characterization of autoimmune serum. Joos and colleagues spotted 18 common
autoantigens onto silane-treated glass slides and nitrocellulose at serial dilutions.
Bound antibodies from minimal amounts of 25 characterized autoimmune serum
samples and ten normal blood donors were titered by using variable amounts of
autoantigen. The autoimmune serum samples were obtained from patients with
autoimmune thyroiditis (Hashimoto’s thyroiditis and Graves’ disease), systemic
lupus erythematosus (SLE), Sjogren’s syndrome (SS), mixed connective tissue
disease (MCTD), scleroderma, polymyositis, systemic vasculitis, and antiphos-
pholipid syndrome. These assays proved to be highly specific and similar in
sensitivity when compared to a standard ELISA format. Further developments
will include optimizing the nature of the autoantigen material to minimize pos-
sible loss of antigenicity and expanding the representation of autoantigens on
the array.
Similarly, Robinson and colleagues have developed a 1152-feature array con-
taining 196 distinct biomolecules representing major autoantigens targeted by
antibodies produced by rheumatic autoimmune disease patients [4]. The autoanti-
gens included hundreds of proteins, peptides, DNA, enzymatic complexes, and
ribonucleoprotein complexes. Examples of autoantigens spotted include Ro52,
Ro60, La, jo-1, Sm-B/B
, U1-70 kD, U1 snRNP-C, topoisomerase 1, pyruvate
dehydrogenase (PDH), and histone H2A. The arrays were characterized using mul-
tiple sera from eight human autoimmune diseases and included SLE, SS, MCTD,
polymyositis, primary biliary cirrhosis, rheumatoid arthritis (RA), and both lim-
ited and diffuse forms of scleroderma. This work demonstrates the feasibility
of using large-scale, fluorescence-based autoantigen microarrays to detect human
autoantibodies with simple protocols and widely available equipment in a low-
cost and low-sample volume format. Some of the potential applications for this
technology include (1) rapid screening for autoantibody specificities to facilitate
diagnosis and treatment, (2) characterization of the specificity, diversity, and epi-
tope spreading of autoantibody responses, (3) determination of isotype subclass
of specific autoantibodies, (4) guiding development and selection of antigen-
specific therapies, and (5) use as a discovery tool to identify novel autoantigens or
epitopes.
Microarrays that simultaneously detect multiple cytokines have been developed
by Huang and colleagues at Emory University [5]. Their method utilizes capture
antibodies spotted onto membranes, incubation with biological samples such as
patient serum, and detection by biotin-conjugated antibodies and enzymatic-coupled
enhanced chemiluminescence. Twenty-eight cytokines were detected using this
4 DNA Microarrays and Related Genomics Techniques

method, including interleukins-1α, 2, 3, 5, 6, 7, 8, 10, 13, and 15; tumor necrosis

factors α, and β; interferon-γ, and others. In addition to detecting multiple cytokines
simultaneously, these assays were shown to be more sensitive than conventional
ELISAs, with broader detection ranges. The ability to readily scale up this approach
to include much larger numbers of cytokines and other proteins will undoubtedly
fuel further development of this powerful tool for studying complex and dynamic
cellular processes such as immune reactions, apoptosis, cell proliferation, and
differentiation.

1.4 DNA AND OLIGONUCLEOTIDE MICROARRAYS

DNA microarrays were first introduced in the mid-1990s [6] and have been the
most widely utilized application of microarray technology. There are two com-
monly available DNA microarray systems. First are the cDNA microarrays fabricated
by robotic spotting of PCR products, derived primarily from the 3
end of genes
and expressed sequence tags (ESTs), onto glass slides — this is the method pop-
ularized by, among others, Dr. Patrick Brown at Stanford and Dr. Louis Staudt
at the NIH [7,8]. The second method uses in situ synthesized oligonucleotide
arrays that are fabricated using photolithographic chemistry on silicon chips —
this is the method used in the proprietary AffymetrixTM system [9] and recently
by NimbleGenTM . A third method involves spotting previously synthesized longer
(40 to 70mer) oligonucleotides on either glass (AmershamTM and AgilentTM ) or
nylon and plastic (clonetechTM and SuperArrayTM ). The data generated using these
systems are highly concordant, as demonstrated in parallel studies of the yeast cell
cycle [10,11]. In the spotted cDNA and long oligo microarray systems, two probes
with different fluorescent tags are hybridized to the same array, one serving as the
experimental condition and the other as a control. The ratio of hybridization between
the two probes is calculated, allowing a quantization of the hybridization signal
for each spot on the array. In this system, the probe is 1st strand cDNA gener-
ated by oligo-dT primed reverse transcription from an RNA sample (for additional
details see http://cmgm.stanford.edu/pbrown/). In the AffymetrixTM system, only
a single labeled probe is used and each gene on the chip is represented by 8 to
10 wild-type 25-mer oligonucletides and the same number of single base mutant
25-mer oligonucleotides synthesized next to one another on the array. Signal intens-
ity and the ratio of specific to nonspecific hybridization allows the generation of
quantitative data regarding gene expression in the sample (for more details see
http://www.affymetrix.com/technology/tech_probe.html).

1.5 TILING ARRAYS

Recently several groups have developed arrays with long stretches of chromo-
somes or whole-genomic sequences probed onto arrays. Potential uses for such
whole-genome arrays include empirical annotation of the transcriptome [12],
Microarray Platforms 5

identification of novel transcripts [13,14], analysis of alternative and cryptic

splicing, characterization of the methylation state of the genome, polymorph-
ism discovery and genotyping, comparative genome hybridization, and genome
resequencing [15]. These arrays have great future potential for studying new
aspects of the genome and providing greater insights into the function of living
organisms.

1.6 DATA ANALYSIS

Microarray analysis is often considered a discovery-based rather than hypothesis-
driven approach [16,17], largely due to the potential for discovering altered expression
of novel genes for which little or no prior information was available to sug-
gest a role in the disease or experimental condition examined. However, high
quality experiments are driven by addressing a scientific question (even if it is
simply — “are there genes that are differentially expressed between a group of
patients and controls?”), consistency in execution of experimental protocols, use
of sample sizes with as many replicates as is feasible, and a plan for statistical
analysis and interpretation of the data. Including statistical expertise during the
early phase of experimental design (i.e., prior to any data collection) is critical,
particularly in the setting of microarray analysis where each experiment can carry
significant cost.

1.7 FUTURE DIRECTIONS

The majority of human diseases undoubtedly involves the complex interplay of many
genes. Although the number and type of genes are not yet known, global assess-
ment of gene expression is a very powerful approach for gaining insight into these
processes. Identification of these genes will certainly contribute to advancing our
understanding of the molecular basis for human diseases and identifying novel thera-
peutic targets. Within a relatively short period of time, the information learned
from the application of microarray technology to address complicated biological
questions has not only met, but often exceeded expectations. Despite their success,
microarray studies are not without their challenges. Continued refinement of these
techniques, including development of improved statistical methods for extracting
information from large datasets and software tools for data processing, manage-
ment, and storage as described in the following chapters of this book, will likely
increase the applicability and general use of these technologies. Additionally, estab-
lishing common standards for the publishing and sharing of microarray generated
data will be important. The applicability of this technology in translational medi-
cine is only beginning to be appreciated and it is likely that microarray technologies
will have a substantial impact on our understanding of human disease now and into
the future.
6 DNA Microarrays and Related Genomics Techniques

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 The establishment and economy of our house were upon the most
regular plan; the former consisted of six apprentices, (including
myself,) and four assistants at very liberal stipends, besides a
nephew of the elder partner, who superintended the whole, and
officiated in the compting-house: there were also several porters,
and other subordinates, for all of whom full employment was found.
Being the junior apprentice, it was my province to polish the
counters, trim the lamps, carry out small parcels, and to perform
other inferior duties; when disengaged from which, I assisted in
waiting on the retail customers, and making myself otherwise useful
behind the counter. We had a plentiful table appropriated for us, to
which we retired in turn during the hours of business; commodious
and airy chambers; and, in short, enjoyed every comfort we could
desire.
For the first month of my probation, I behaved extremely well,
and by my quickness and assiduity, gained the good opinion of my
employers, who wrote of me in the most favourable terms to my
friends in S⸺shire; nor did my expenses exceed my allowance for
pocket-money, which was fully adequate to every rational
enjoyment.
Among my fellow apprentices, was a young man named King,
some years older than myself, with whom, from a similarity of
sentiments, I formed a close intimacy. He was of an excellent
disposition, but a great lover of pleasure; and as his servitude was
far advanced, and his prospects peculiarly flattering, he was under
very little restraint, but gave the rein to his passion for dissipation.
His expenses were profuse, but whether he indulged in them at the
expense of his probity, I could never ascertain. He soon introduced
me to several young men of his own stamp, and I became in a short
time as great a rake as the best of them: nor was our conversation
confined to our own sex; scarcely a night passing without our
visiting one or other of those houses consecrated to the Cyprian
goddess, with which the town of Liverpool abounds. In such a
course of life, it is not likely that I could submit to limited hours; my
companion and I seldom returned home before midnight, and
sometimes not till the ensuing morning. Though we took measures
to keep this from the ears of our employers, it could not fail to be
known in time; and the consequence was, a strong but tender
remonstrance on my imprudence, which much affected me at the
moment; but the impression was transitory, and soon effaced. I
plunged deeper and deeper in the vortex of folly and dissipation,
until I was obliged to have recourse for advice to the Æsculapius of
Gilead-house.
This irregular mode of life had borne hard on my finances, but I
had not, as yet, had recourse to fraud or peculation. I was liberally
supplied by my relations, on leaving S⸺, and had received my first
quarterly allowance; but an event, which soon followed, tempted me
to the first breach of confidence and integrity.
I had in my youth been passionately fond of Cocking, a sport for
which the county of S⸺ has been always famed; and though so
young, I had constantly kept several cocks at walk, unknown to my
parents; so that I had acquired a considerable share of experience
and knowledge on the subject. One day, when I was sent with some
muslins to wait on a lady in the environs of Liverpool, near the
Canal, I accidentally passed a cockpit, where a great crowd was
assembled; and I understood that a grand Main was about to
commence. Elated at this pleasing intelligence, I hastened to
execute my commission; and returning to the house, entered it, and
leaving my wrapper of goods in care of the landlady, I ascended to
the pit, and took my seat. The company was, as usual, of a motley
description, but there were many genteel persons. I ventured a few
trifling bets at first with various success; but at length an
opportunity offering, which I considered as next to a certainty, I laid
the odds to a large amount, flattering myself that by this stroke of
judgment, I should be enabled to figure away with increased eclat
among my gay companions. After I had so done, greater odds were
still vociferated, but in a moment the scene was changed! the fallen
cock, in the agonies of death, made a desperate effort, and rising for
a moment, cut the throat of his antagonist, who was standing over
him, in the act of crowing with exultation on his victory! The latter
immediately fell, choked with the effusion of blood, nor did the victor
survive him many moments. The whole pit resounded with
acclamations, and the discord which ensued beggars description. I
was not the only sufferer by this revolution of fortune; many others
had laid higher odds than myself, and to a much greater amount. I
was soon surrounded by my creditors, to whom I disbursed every
shilling I had about me, among which were some pounds I had just
received from the lady for goods, and for which I had given her a
receipt. I was still something deficient, for which I pledged my
honour to one of the parties, giving my address, and promising
payment on an early day. I now returned home, filled with remorse
and shame; but as the first false step of a young person insensibly
leads to another, I added to my guilt by concealing the affair from
my employers, and directed them to book the articles the lady had
selected. I had a degree of false shame about me, which rendered
me incapable of confessing the truth and promising amendment, or
all might still have been well. In the evening I had recourse to the
bottle to drown my chagrin; and I determined to purloin a certain
sum every day, in the course of my attendance on retail customers
until I had liquidated my debt of honour! Then, I vowed to stop and
reform. Delusive idea! how little did I then know my own weakness,
or the futility of such resolutions in a young mind! And who, that
once begins a career of vice, can say to himself, “Thus far will I go,
and no farther?” After I had discharged my engagement, I found a
small sum must be raised for pocket-money, and other exigencies,
as it would be above two months before I could expect a remittance.
I therefore continued my peculation, and at length my evil genius
suggested to me, that I might, by venturing a small sum, become
more fortunate at the cockpit, and repair the loss I had sustained; as
miracles don’t happen every day, and the odds must win in the long
run. Thus I argued with myself; and, fatally for me, I tried the
experiment.
From this moment I never missed a day’s fighting at the cockpit;
and when sent on business which required my speedy return, I could
not tear myself from the spot, but frequently staid out several hours,
and afterwards forged a lie to account for my delay. I sometimes
came off a winner, but as I was not then acquainted with the art of
hedging, by which the knowing ones commonly save themselves, I
was sure to be a loser at every week’s end.
I managed matters so well, indeed, that my frequent secretions
from the till, were not discovered, however they might be suspected.
The extensive trade of the shop rendered it next to impossible; and
what I abstracted, was a trifle compared to the gross receipts of the
day.
My continued misconduct became now the subject of frequent
remonstrances on the part of Mr. Parker the resident partner; which
not having the desired effect, that gentleman wrote to my friends,
informing them in general terms, that I had unhappily formed
improper connexions, and that my late levity of conduct rendered
me unfit to be received into their house; therefore, desiring I might
be recalled without delay. Mr. Parker concluded with a remark, which
I shall never forget, and which was peculiarly gratifying to my
grandfather’s (perhaps too partial) feelings: after expatiating on my
general capacity for business, he added “his smartness and activity
are really wonderful.” This letter produced a speedy answer, in
consequence of which, I was directed to hasten my departure, which
took place in a few days, Mr. Parker giving me a great deal of
wholesome advice at parting; observing that although it was not in
his power to charge me with any direct criminality, my inconsiderate
behaviour, and the continued excesses of my conduct, left but too
much room for unfavourable conjectures.
Behold me now returned to my grandfather, after an absence of
nearly five months; and this excursion may be called my first
entrance into life. I could not help blushing at the consciousness of
my own unworthiness; but the blind partiality of my dear parents,
induced them to believe me less culpable than I really was; and to
listen readily to any thing I had to offer in palliation of my errors.
 CHAPTER IV.
I prevail on my Grandfather to let me visit London.—Am provided with Letters of
Recommendation.—Received into the Office of a respectable Attorney, my
Kinsman.—Quit that Employment, and engage with a wholesale Stationer.—
Obtain Clothes, &c., on credit, without any intention of paying for them.—Bilk
my Lodgings repeatedly.—Return to the Law.—Obliged to live by my Wits.—
Become a Hackney Writer.—Resort nightly to the Blue Lion.

Shortly after my return from Liverpool, finding the narrow limits of

a small market-town too circumscribed for my active disposition; and
nothing having been determined on, as to my future disposal, I
entreated my parents to let me go to London, which I had always
considered as the grand field for talents of every description; and
where I doubted not of forwarding myself, by the exercise of those I
possessed. They at length consented, and after the necessary
preparations, I set out, receiving the blessings and prayers of these
indulgent benefactors, and moderately supplied with money for my
reasonable necessities.
My grandfather had of course many acquaintances in London, of
his own profession, to several of whom he furnished me with letters
of recommendation; it being his wish, that I should by a few months’
practice in a law office, ascertain how far I was disposed to be
articled for the usual period, necessary to procure my admission. I
had always been prepossessed in favour of this profession, which I
knew from my reading and experience, to abound in variety, and, if I
may be allowed the phrase, in pleasing perplexity.
On my arrival in town, I waited first on a relation of our family, Mr.
P⸺, a young man lately established in the profession, and received
as a partner by Mr. M⸺, a gentleman of large fortune, and
extensive practice, who would, it was probable, in a few years’ time,
retire from business in favour of my cousin.
 The latter received me very kindly, and without hesitation, offered
to employ me (with his partner’s concurrence) as copying clerk, at a
weekly salary, which to a youth like me, was of course but small.
As I conceived this offer too advantageous to be rejected, I
thankfully embraced it, and immediately entered on my function in
Lincoln’s-Inn New Square.
As I had no motives of affection to attract me to my father’s
house, I determined on becoming master of my own conduct; and
accordingly took a neat private lodging, and regulated my mode of
life conformably to the state of my finances. I breakfasted at home,
dined at a tavern or genteel eating-house, and in the evening took
my tea and read the papers at a coffee-house: after which I
sometimes passed the night in reading at home, but most commonly
went to one of the theatres at half-price, where I gratified my violent
passion for the drama, which at once improved my understanding
and amused my mind.
This course of life, though it rapidly weakened my purse, was
rational, compared to that which I soon after led, and I might have
supported it with credit, by the indulgence of my friends, for a
considerable time. As the business of our office was of a various
nature, I soon gained great experience in the different branches of
common law, and conveyancing: I became by practice, an expert
and correct copyist; and I delighted much in studying the most
approved law books, reading reports of cases, &c. I became familiar
with Burn’s Justice, and soon gained a pretty correct knowledge of
the criminal law, and of those minute points, of which an able
counsel or attorney can avail himself, in order to rescue a client from
the claws of justice, howsoever glaring and palpable his guilt may
be. Such is the glorious uncertainty of the English law! Little did I
then conceive how useful this knowledge might one day prove to
myself; of which more hereafter. I merely pursued this intricate
study by way of exercising my ingenuity and gratifying my curiosity.
I must here inform the reader, that these various pursuits and
studies were not confined to the short term of my continuance in the
employ of which I am now writing. After I quitted the office of
Messrs. M⸺ and P⸺, and during the course of a wild and
dissipated life, they formed one of my favourite domestic
amusements; as did in fact reading in general: and throughout the
most profligate stages of my future career, which I shall hereafter
narrate, a portion of my time was always devoted to the perusal of
books, and a part of my money, however hardly or dishonestly
obtained, to the purchase of them: and to this moment I still
consider them the most valuable property a man of my disposition
can possess.
About three months after my arrival in town, I began to grow less
regular in my manner of life; my expenses increased; and I became
negligent of the office hours. I had contracted an intimacy with
several young persons of both sexes, which unavoidably engaged
me in a course of expensive dissipation, to which my means were
inadequate. Though I occasionally derived small supplies from my
grandfather, yet as he had no idea of the extent of my expenditure,
they were comparatively trifling. Mr. P⸺ finding I was not to be
reclaimed by advice, and having no authority to use any other
means, at length for the credit of his office, and for fear I should
contaminate the other clerks, gave me a formal, though civil
dismissal, which I received without surprise, as I knew I had long
deserved it.
I was now tired of the law, or rather of the restraint I was under,
in an office of that description; but still I found it necessary to have
some employment, as well to amuse me, as to contribute to my
support.
I therefore engaged myself as assistant in the warehouse of
Messrs. Key and Sons, wholesale stationers in Abchurch-lane,
Lombard-street, at a weekly salary of one guinea; which was more
by half than I had received from my last employers, and equal to the
pay of an experienced clerk in an attorney’s office at that time of
day. This situation I obtained through the interest of one of my
fellow-rakes, the son of a substantial citizen, who roundly vouched
for my integrity and diligent conduct. My employment here was to
superintend the packing of goods for exportation and home
consumption; to write invoices; copy letters; ship goods at the
Custom-house, &c. I continued, however, but a short time in this
situation; confinement to the east end of the town prevented my
associating with my old acquaintances, who chiefly dwelt in the
environs of Covent-garden, or the purlieus of Drury-lane.
During an abode of ten months in London, as I was frequently
pushed for money, I availed myself of a genteel appearance, and
pretty good address, and taking advantage of the credulity of several
tradesmen in the neighbourhood, I ordered wearing apparel of
various kinds, and sometimes other goods, upon credit, without
much concern about the day of payment; however I always took
care to procure a bill of parcels with the articles, which precluded
any charge of fraud, and left the matter at the worst but a debt
contracted; for which, being a minor, I knew I could not be arrested.
This was my first deviation from honesty, since I left Liverpool. I was
also frequently obliged to change my lodgings; and, as payment of
my rent would have required ready money, for which I had so many
other uses, I commonly decamped under favour of the night, having
previously removed my effects by various stratagems. As I was
ashamed to let my grandfather know the true state of my affairs,
and as I really grieved at the expenses I had already caused him,
which I knew had much inconvenienced him, I forbore at last, to
trouble him for remittances; but falsely assured him that I was doing
well, and enabled to live upon the profits of my industry. I desired
he would abandon the idea of articling me to the law, as the
expenses attending admission had of late years been so much
increased by stamp duties; and as I could, if I continued the study,
at a future period, practise under the sanction of another person’s
name; a custom then very prevalent, though irregular. By these
assurances I quieted the good old man, and silenced any inquiries
my friends might have instituted respecting me; as I now really
wished to continue free from all restraint upon my person or actions,
and foolishly flattered myself that I should by some lucky event,
ultimately secure the means of independence. These conjectures
were, however, perfectly vague, and proceeding from no fixed idea
whatever.
On quitting my city employment, I returned to the law, for which I
still retained a partiality; and obtained a more liberal salary than
before in an office equally respectable. Indeed I was now become
more useful, and had improved much both in person and address
since my arrival in town.
I was still frequently reduced to pecuniary straits, and obliged to
have recourse to various expedients, known only to men of the
town, for my support: some of them indeed were bordering on
dishonesty, and none of them very honourable. But to describe them
individually, is impossible, and a man who lives by his wits, as the
phrase is, will assure you, if called to account, that he really could
not for his life, tell by what distinct means he makes out a living.
As I now wrote uncommonly fast, I quitted the station of a weekly
clerk, and obtained writings to copy by the sheet, from the law
stationers, by which I could earn considerably more money; and in
this employment I continued to labour diligently for several hours
every day, and sometimes half the night.
When I had a mind to relax from this occupation, and particularly
if my finances were at a low ebb, I frequently resorted to the Blue
Lion, in Gray’s-Inn-Lane, a house noted for selling fine ale, and
crowded every night with a motley assemblage of visiters, among
whom were many thieves, sharpers and other desperate characters,
with their doxies. I was introduced to this house (from which
hundreds of young persons may date their ruin) by a fellow-clerk,
who appeared to have a personal intimacy with most of these
obnoxious persons; however, though I listened eagerly to their
conversation, (part of which was then unintelligible to me), and
fancied them people of uncommon spirit, I was not yet sufficiently
depraved to cultivate their acquaintance; but sat with a pipe in my
mouth, enveloped in smoke, ruminating like a philosopher on the
various characters who tread the great stage of life, and felt a sort
of secret presentiment, that I was myself born to undergo a more
than common share of vicissitudes and disappointments. How far
these ideas were well grounded, the reader will judge when he has
perused this narrative, of which I shall here close the fourth Chapter.
 CHAPTER V.
The Trip to Portsmouth.

During my nightly resorts to the Blue Lion, in Gray’s-Inn-Lane, I

one evening fell into conversation, at that house, with a young man
of decent appearance, a few years older than myself. We were so
mutually pleased with each other, that at parting, an appointment to
breakfast together the next morning was the consequence, and on
this second meeting, our intimacy so far increased, that we began to
explain our respective situations in life to each other. My new
acquaintance, whose name was D⸺, informed me that he had
lately quitted His Majesty’s Ship Montague, of 74 guns, on board of
which he had served as Steward to the Commander, Captain
Patterson. That having lived freely for some months on shore, he
had now spent all his money, and was so much reduced, that he
really knew not how to subsist any longer, and concluded with
expressing his intention to proceed immediately to Portsmouth, in
the hope of obtaining a similar birth to his former one. I assured him
that I was, like himself, so much reduced, as to find the utmost
difficulty in existing at all, and that if he would permit me to join my
fortune to his, I should without hesitation feel happy in the
opportunity of accompanying him to Portsmouth in quest of some
employment. He declared that nothing would afford him greater
pleasure, and it was agreed that we should, each of us without
delay, convert our spare apparel, &c., into cash, and set out the very
next day. I accordingly lost no time in the needful preparations, and
having bilked my landlord, with whom I was some few weeks in
arrears, I met D⸺, the same evening, and after indulging in a
farewell cruize, which sensibly diminished our slender finances, we
took a few hours’ repose, and at eight o’clock the next morning,
commenced our peregrination.
 On a comparison of our personal effects, it appeared that we
possessed between us about twenty shillings in money, and had
each a spare shirt, neck-cloth and hose, in a bundle, which
(traveller-like) we carried across our shoulders on a good oak-stick.
We were both decently clad, in good health, and in high spirits
notwithstanding our poverty. We agreed that to save expenses, we
should perform the journey on foot, (being seventy-two miles) and
we calculated that with frugality, we should be enabled to defray the
contingent charges for diet and lodging on the road. It soon
appeared, however, that though good economists in theory, we were
not so in practice; for as we both loved good cheer, and the severity
of the weather required a suitable fortification, we had expended
half our stock by the time we got to Kingston, our first stage, and
only twelve miles from town. Here we put up at a public-house,
called The Eight Bells, and having enjoyed a good dinner, which was
succeeded by some excellent mulled elder wine of the landlady’s
own composition, and which was peculiarly adapted to the season of
the year, we liked our quarters so well that we spent not only the
night, but half the next day in the enjoyments of repose, eating,
drinking, and smoking, before it occurred to either of us that every
item of those said enjoyments was recorded in chalk by the hostess,
and would inevitably be consolidated in the shape of a bill, which we
must discharge before a clearance could be obtained for the next
port. Having at length recollected ourselves, and called “to pay,” our
reckoning amounted to three or four shillings more than we
possessed in our common purse. As it was impossible to think of
proceeding any farther without recruiting, we were now in no small
consternation. Bilking the landlady was out of the question, for we
had given our bundles into her charge on first entering the house,
and their contents were not only indispensably necessary to us, but
also worth more than the sum required to release them. In this
dilemma, a sudden thought struck me. Calling for pen, ink, and
paper, I told my companion I had a scheme in my head for raising a
supply, but would not impart it until I had tried its success. I then
drew up a sort of Memorial to the following effect: “To the Ladies
and Gentlemen of Kingston.—The writer hereof, a young man of
respectable family, and good education, having by a series of
misfortunes, been reduced to the greatest distress, is now on his
way to Portsmouth, in hopes of procuring a situation in the navy; but
being destitute of money for his present support, humbly solicits
your charitable assistance towards enabling him to pursue his
journey. To a noble mind, the pleasure of doing a good action is its
own reward. The smallest donations will be gratefully received, and
any lady or gentleman inclined to relieve the writer is earnestly
requested to subscribe his or her name hereto.”—Having completed
this production, I desired my friend to wait patiently for my return,
and assured him I doubted not of bringing speedy relief. I now set
out on my expedition, and immediately waited on Mr. Mayor, who
was a grocer, but in this first essay I was unsuccessful. His worship
declared he never encouraged applications of this sort from
strangers; and desired me to go about my business. I, however, took
the liberty of subscribing his name to my memorial, by way of
sanction, and gave his charity credit for a donation of five shillings.
Young as I was at that time, I well knew that example, in matters of
this kind, goes a great way; and that many persons, without a grain
of christian benevolence in their composition, will give liberally from
motives of ostentation, when they see that their neighbours have
already contributed, and that their own names and donations will
also be made public. I experienced the truth of this notion, for I was
successful in almost every application I afterwards made. Having
visited a number of genteel houses, with various success, I was on
the point of returning, to impart my good luck to my companion,
when coming to a very handsome mansion-house in the suburbs of
the town, I thought I ought not to omit calling, and a person at that
moment passing by, I inquired whose residence it was, and which
was the entrance to the premises; for the house was situated in the
midst of a spacious pleasure-ground, remote from the high road,
and it being quite dusk, I had not observed any avenue by which I
could gain access to it. I was informed that it was the residence of
Lady W⸺, that a little further on, I should perceive a door in the
brick wall, which extended along the road-side, and that if I entered
at that door, and proceeded in a strait direction, I should arrive at
the servants’ hall; but my informer cautioned me to keep close to
another wall on my left hand, which divided this avenue from the
lawn in front of the mansion, because there was a very large and
fierce dog at the upper end, but which, being chained up, could not
reach me, if I followed the above directions. I thanked this obliging
person, and immediately proceeded to the door described, which I
entered, and walked cautiously, and not without some fear, by the
wall-side, till I perceived by the lights in the kitchen, and out-offices,
that I was near the premises.
It was now very dark, and I was carefully exploring my way, my
mind full of apprehensions at the thought of this terrible dog; when
lo! at that instant, to my inexpressible consternation, the ferocious
animal made a spring at me, and I gave myself up for dead.
However, though he was certainly within a yard of me, he did me no
mischief, but my alarm was so great, that without knowing how, or
where to fly for refuge, I ran precipitately from the spot, and when I
recovered from the fright, found myself in the pleasure-ground in
front of the mansion-house. It appeared that I had, without
knowing, escaped through a door in the wall, which was open on my
left hand at the moment I was alarmed by the dog. I was now more
at a loss than ever, for I knew of no way to get out of the pleasure-
ground, except by the aforesaid door, and fear of the dog prevented
my attempting that passage. After wandering about for a few
minutes, I approached the mansion, and going up to one of the
parlour windows, which were very large, and on a level with the
terrace before the house, I applied my eye to the glass, and
discovered through an aperture in the inside shutters, a numerous
and splendid party of ladies and gentlemen at dinner. Having
considered a moment, I determined on a very bold step, as I saw no
alternative but remaining all night in the open air, exposed to the
inclemency of the weather. Taking advantage of a pause in the
company’s conversation, I tapped with my finger at the window, and
immediately the whole party were struck with wonder. In the midst
of their surprise I repeated my knock, and then, after several voices
exclaiming, “Good God! there is certainly somebody at the window,”
&c., a gentleman rose from the table, and advancing towards me,
opened first the shutters, and then the window itself, which might, in
fact, be called a pair of folding doors, and these being thrown back,
I walked in with the most respectful air I could assume, and
presented myself to the astonished company. Having bowed twice or
thrice, and given time for their alarm to subside, I began to make
my speech.
Apologizing for my presumptuous intrusion, I stated in a concise
manner, the fright I had endured, from the dog, my embarrassment
at not being able to find means of egress from the pleasure-ground,
and my having consequently taken the liberty of knocking at the
window. I then presented my memorial, which was read in turn by
most of the company, each of whom surveyed me with evident
surprise. Having answered such queries as they thought proper to
put to me, I was desired by the lady of the house to withdraw to the
kitchen for a short time, and a servant was ordered to attend me
thither. Here I had my story to repeat for the information of the
domestics, who laughed heartily at the adventure of the dog, but
afterwards seriously assured me, that had the animal not been
chained, or had I approached within his reach, he would inevitably
have torn me to pieces. The parlour dinner being over, and the
dishes brought out, I was desired to fall to, and being really hungry I
wanted no pressing, but selected from the variety of good things on
the table a very fine buttock of beef, on which substantial fare I
made a sumptuous meal. There was no scarcity of good malt liquor,
and Lady W⸺ very kindly sent me out a pint of red port, with a
particular injunction (which by-the-by was unnecessary,) that I
should eat and drink heartily.
At length I was summoned to attend the company in the parlour,
and her ladyship then expressing her concern for my misfortunes,
and her anxious hope that I should speedily find an end to them,
presented me with half-a-guinea. The rest of the party also said
many handsome things, and the majority of them contributed to my
relief. In addition to these favours, one of the gentlemen, at the
particular request of Lady W⸺, took the trouble to write a letter in
my behalf to the captain of a man-of-war, supposed to be then lying
at Portsmouth, entreating him to give me an appointment under
him. Her Ladyship, after obliging me to take another glass of wine,
and repeating her sorrow for my distress, advised me to lose no time
in prosecuting my journey, ordered a servant to conduct me to the
door at which I had first entered her premises, and I took a
respectful leave of this truly benevolent party.
Returning to the Eight Bells, I imparted my adventures to my
friend, who was, of course, much pleased at my success, for I had
realized between four and five pounds. I found this begging scheme
so productive, that I was in no hurry to pursue the Portsmouth
speculation, and as we were both satisfied with our present
quarters, it was agreed that we should continue a few days longer in
Kingston, in which time I proposed to follow up my success by
making a regular circuit among the inhabitants; and I, in fact,
determined to levy similar contributions in every town which lay in
our route.
It is to be observed, that this idea of raising money was perfectly
original in me, for at that time I had never heard of such a practice,
but have since discovered that it is a very common expedient, and is
called by those persons who live by such impositions, “the Letter
Racket.”
The following day I again sallied forth, and met with equal
success, visiting not only the houses of private persons, but even the
respectable shopkeepers, &c.; and I may here state, once for all,
that in the course of this, as well as my subsequent speculations of
the same nature, I met with various receptions, according to the
charitable or churlish dispositions of the people to whom I applied.
Many pitied my case and cheerfully relieved me. Others expressed
equal commiseration, but declined giving any thing, either because
“they never encouraged beggars,” or “they had poor enough of their
own to maintain.” Some invited me into their parlours, treated me
with excessive politeness, and obliged me to take refreshment at
their own tables; and where there were any young ladies in the
family, I was an object of particular solicitude, and the recital of my
misfortunes drew many a sigh from their tender bosoms. Others
desired me, like the Mayor of Kingston, to go about my business,
and hinted that I ought to be sent to the house of correction as a
vagrant. Sometimes the servants who admitted me refused to
present my memorial, declaring they had strict orders from their
masters or mistresses never to trouble them on such an occasion.
The donations I commonly received, were from one shilling to five;
sometimes, but rarely, I was presented with gold, particularly at the
seats of the nobility and gentry, all which, laying within a short
distance of the road I travelled, I made a point of calling at; and for
my information on this subject, I provided myself with a
comprehensive book of roads, in which those objects are correctly
laid down. Some truly charitable persons, but whose means were
limited, relieved me with sixpence, and of course I was bound to
accept such a trifle with as much appearance of thankfulness, as I
would a larger sum; and frequently when I called at a farm house by
the road-side, I have been compelled to take some cold meat, or
other eatables, which I afterwards bestowed upon the first more
needy beggar I met on my way. It was my custom in general, to
travel on foot, making short stages, and putting up at a good inn in
every town I entered, where I lived upon the best during my stay,
and associated with London riders, and other respectable guests.
When tired of walking, I availed myself of a passing stage-coach, or
return post-chaise, and my only equipage was a spare shirt,
handkerchief, &c., which, with my book of roads, I carried in a small
bundle under my arm.
To resume my narrative, I returned from my second day’s
excursion about dusk in the evening, and entering the Eight Bells,
was surprised at not finding my companion within. On inquiring of
the landlady, the good woman informed me, with some degree of
terror, that two of the town constables had been in search of me;
and kindly exhorted me, if I was conscious of having done wrong, to
make the best of my way from Kingston, as she expected them to
call again every minute, adding that she believed my friend was
gone out in quest of me. I smiled at the landlady’s fears, and
thanking her for her friendly advice, assured her I was unconscious
of any crime, and should willingly face the officers, come when they
might. I then desired to have some tea, which she set about
preparing, during which interval my fellow-traveller came in. I briefly
concerted with him the part we should respectively act, in case of
our being called upon to give an account of ourselves, though I had
no conception on what grounds I was to be apprehended. We now
proceeded to take our tea, but before we had half finished it, the
constables entered the room, and informed me they had orders to
carry me before the magistrates, who were then assembled in the
town-hall; and that the young man, who they understood to be my
comrade, must also accompany me. I answered, I would with
pleasure attend them, but requested time to finish our repast, which
they consented to allow us; and we then proceeded all together to
the town-hall, the officers carrying our two bundles, which they had
obtained from the landlady by order of the justices. Being brought
before the bench, I immediately recognised among the persons in
attendance, one of the gentlemen I had seen at Lady W⸺’s dinner
party, and the very same who had written the letter of
recommendation to Captain ⸺. This gentleman now stated to the
magistrates the circumstance of my coming to Lady W⸺’s house
the preceding evening, the extraordinary manner in which I had
made my appearance, and the reception I met with; and farther
stated, that the ensuing morning, it was discovered that a part of
the fence enclosing the pleasure-ground had been broken, and other
damage done on the premises, which led to a suspicion that I might
be an impostor, who had a design to rob the house, or do some
other mischief, and that he, therefore, on the part of Lady W⸺,
wished me to be examined by their worships, and obliged to give
some further account of myself.
Though the suspicions of Lady W. and her friends appeared to
have been very weakly founded, and their idea to be utterly
extravagant, the justices entered seriously into the spirit of the
business, and ordering us to be set apart from each other,
proceeded to interrogate us strictly as to our professions, business in
Kingston, the objects of our journey, &c. I, for my part, adhered to
the story I had before framed, which it is needless to repeat to the
reader, being devoid of interest, and as it was for the most part
utterly false, I cannot now minutely recollect it. My companion’s
account of himself was nearer the truth. He stated that he had been
an officer’s servant in the navy, and was now on his way to
Portsmouth in quest of a similar situation, but denied any further
knowledge of me than joining me by accident on the road, and as I
was bound to the same place, we had agreed to keep each other
company. Our bundles being next inspected, were found to contain
nothing of a suspicious nature, except a number of pawnbrokers’
duplicates, and these we respectively accounted for as relating to
wearing apparel, &c., which our pecuniary distress had obliged us to
pledge. This part of the story was indeed strictly true, and in my
case corroborated, and gave an air of probability to my account.
Nothing appearing to warrant the detention of my companion, he
was discharged, but with a positive order to quit the town instantly.
As to myself, having mentioned, in answer to a query from one of
the magistrates, the name of a certain gentleman, in London, who
knew me and my family, his worship declared he was himself
intimately acquainted with that gentleman, and as he was going to
town the next day, he should make a point of referring to him as to
the truth of my assertion. He therefore ordered a constable to detain
me in custody until his return from London. I was accordingly
confined in the officer’s house, where I had every necessary
accommodation, for the expenses of which the magistrate himself
very kindly became accountable. My companion visited me the same
evening, prior to his departure, and I divided with him the money I
had acquired. It was his intention to proceed direct to Portsmouth,
and I promised, as soon as I was liberated to follow him, taking a
direction whereby to find him on my arrival.
The ensuing evening the magistrate returned from town, and
immediately sent for me to his own house. Being brought before his
worship, who was a brewer, named Sidebottom, I found him seated,
not on the chair of justice, but on a stool in his accompting-house.
The old gentleman began by remarking with some severity, on the
impropriety and turpitude of my conduct, in withdrawing myself from
the protection of my respectable friends, and traversing the country
under such debasing circumstances. He said he had understood
from his friend Mr. Howell, that my relations were both able and
willing to take care of my fortunes. After a long expostulation to the
same effect, his worship continued: “Perhaps you are not aware that
it is in my power to commit you to bridewell for a month for begging
without a pass.” Then raising his voice and brandishing his pen, he
exclaimed with a very pompous air, “Young man, I shall certainly⸺
discharge you.” The two latter words relieved me from a momentary
anxiety; for when his worship paused, I certainly expected he would
add “commit you.” He proceeded, “but, on the express condition that
you immediately quit this town, and return to your friends. If you are
found hereabouts after this night, expect to be severely dealt with.” I
gladly accepted these terms, and assuring the old gentleman of my
compliance, took a respectful leave, his worship giving me a little
more wholesome advice, and generously presenting me with half-a-
crown, as he said, to carry me to London.
Being now discharged, I repaired to the Eight Bells, where I
settled with my landlady, and learnt from her that my companion
had left Kingston the preceding night at a late hour, by the
Portsmouth coach. I slept this night at the Eight Bells, and the next
morning returned to London, where on my arrival I found myself in
possession of above five pounds, after all the impediments I had
met with.
As I made a point of keeping my word with my late fellow-
traveller, I determined on having one jovial evening in town, and
resuming my journey the next day, by the same route as before,
notwithstanding Justice Sidebottom’s prohibition. As to presenting
myself to Mr. Howell, or going near any of my friends, nothing was
more foreign to my thoughts.
 The following morning, I accordingly set out on my second
expedition by the Kingston stage, and arriving at that town, took up
my lodging at a public-house in the suburbs. Notwithstanding the
check I had before met with, I was so hardy as to stop three days in
Kingston, during which I levied contributions on most of the
inhabitants, taking care to avoid that part of the town I had before
visited, and I was so lucky as to meet with no interruption to my
success. From Kingston I proceeded to Guildford, taking in my way,
Cobham, Ripley, and the intervening villages; from Guildford to
Godalming, thence to Liphook, where I altered my course, and took
a circuitous route by the way of Chichester, to Portsmouth. This
journey occupied about a fortnight, and arriving at Portsmouth, I
soon found my late companion, D⸺, who was lodged at the house
of an acquaintance, and not having yet obtained a birth on ship-
board, had engaged himself as an occasional clerk and assistant to a
tradesman in the town. I myself hired a small apartment in St.
Mary’s street, intending to remain a week or two in Portsmouth,
where I had never before been, and to enjoy such amusements as
the place afforded, as long as my money lasted. As my friend D⸺
appeared to have dropped the idea of going on ship-board, I
became also careless on that subject, particularly as the motive
which gave rise to that design (namely, poverty) no longer existed,
for I was now possessed, after all the expenses of travelling, &c., of
about fifteen pounds.
I spent several days in viewing the town, dock-yard, fleet, &c., and
in the evening generally visited the theatre, which was then open.
About a week after my arrival, reading the Portsmouth weekly paper,
I saw an advertisement for a clerk; and, as I found that some means
of subsistence would soon become necessary, and it was indifferent
to me in what part of the kingdom I sojourned, I determined to offer
myself. Inquiring, as directed, of Mottley, the bookseller, on the
Parade, I was referred to Moses Greetham, Esq., judge-advocate,
also an attorney of the first eminence. That gentleman, without
hesitation, engaged me at a guinea a week, and I next morning
commenced my attendance at his office in High-street. About the
same time, my companion D⸺ suddenly quitted the town, without
acquainting me of his intention; and I was informed, on inquiry, that
he had gone to Southampton, but on what speculation I never
learnt, nor did I ever afterwards see or hear more of him.
The nature of my new employment was very agreeable and full of
variety. Besides Mr. Greetham’s public duties on courts-martial, and
his general practice as a solicitor, he had a great deal of business
relating to prizes, their condemnation, sale, proceeds, &c.; and on
these occasions all the parties concerned met in a spacious room at
the Crown inn, where an elegant dinner, with wines in abundance,
was invariably provided at the expense of the clients. These dinners
occurred three or four times every week, and as they were
afterwards succeeded by coffee, &c., the expenses of living, to
myself and the other clerk of Mr. Greetham, who (as well as the
latter gentleman), always partook of the entertainment, were
considerably diminished. During intervals, between dinner, tea, &c.,
we retired to an adjoining room, where an hour or two was devoted
to writing depositions, examining ships’ papers, and other matters
relative to the cause in question. This was, in fact, one of the best
situations I ever met with, and, from its respectability, would, no
doubt, had I continued steady, have ultimately led to prosperity and
independence; but my evil genius interposed to ruin this, as well as
all my former hopes of happiness.
The circumstance which occasioned my losing this place was
indeed very trivial, and hardly amounted to a crime. Among the
number of persons with whom we transacted business, was a native
of Holland, who acted in many cases as an interpreter to Mr.
Greetham, on the part of those concerned in prize causes. This man
being an original, both in person and manners, was an inexhaustible
subject of laughter to myself and fellow-clerk, who was much older
than me, and we omitted no opportunity of indulging in mirth at his
expense. As he was not possessed of much penetration and was of a
very placid disposition, he was commonly insensible of our jokes, or,
at least, took them in good part; but one afternoon, when we were
all three taking coffee together, the Dutchman rising from his seat on
some occasion, with a cup of boiling hot coffee in his hand, my
brother-clerk, who was just then in a merry vein, winked at me to
withdraw the interpreter’s chair. I obeyed the signal with alacrity,
and the poor fellow, attempting to resume his seat, was in a
moment thrown keel upwards, and, as he fell, received the boiling
fluid full in his face. I was so unfeelingly mischievous as to laugh
immoderately at the pain and embarrassment of the poor Dutchman,
who certainly cut a most ridiculous figure; but my fellow-clerk, who
possessed more prudence than myself, restrained his mirth, and,
with a serious air, affected to condole with the sufferer on his
misfortune. He even carried his hypocrisy so far as to express
displeasure at the mischievous trick I had played. I felt much
indignation at his duplicity of conduct, and regretted, when too late,
that I had suffered myself to be made the dupe of his treachery; for
I was afterwards convinced that he was jealous of my abilities, and
feared I should supplant him in the favour of Mr. Greetham. The
consequence of this injudicious frolic was, that the interpreter
complained to my employer, who the next morning rebuked me
severely for behaving with such unbecoming freedom towards a
respectable man in his immediate service; and, observing that such
levity of conduct rendered me unfit for his office, Mr. Greetham
presented me with a guinea, although this was only the second day
of the week, and declared he had no further occasion for me.
Being now once more disengaged, I began to think of returning to
London; but, having still a little money left, I continued about a
week longer in Portsmouth, during which time I took up my lodging
at the Blue Posts’ tavern, in Point-street, where I lived in the most
sumptuous manner, drinking my wine freely, and pretending that I
was waiting for the departure of the East India fleet, in which I was
going out a passenger, and that I expected my baggage from town
in a few days. I had, while in the service of Mr. Greetham, made
several efforts to obtain clothes, &c., upon the credit of my living
with that gentleman; but I found the Portsmouth tradesmen too
wary to be imposed upon, and only succeeded to a very trifling
amount. I had, however, bilked my late lodging in St. Mary’s street,
and intended playing the same trick at the Blue Posts. Finding, at
length, that the landlord and waiters viewed me with scrutinizing
eyes, and appeared tacitly to ask for payment of my reckoning, I left
the coffee-room one evening after dinner, on pretence of going to
the play, and immediately quitted the town, taking the road to
Petersfield on foot.
On arriving at the latter place, which I had omitted in my journey
downwards, by striking into the Chichester road at Liphook, I levied
a small contribution, under the old pretext of going to Portsmouth,
&c., and then pursued my course to London, where I arrived in good
spirits, after an absence of eight weeks, with about ten pounds in
my pocket, having lived well during my expedition, and enjoyed a
variety of pleasant entertainment.
 CHAPTER VI.
Still much embarrassed to support Appearances.—Meet with the Surgeon of a
Frigate.—Our Conversation and its Result.—Negotiation set on foot with the
Captain.—I obtain an Appointment as Midshipman.—Fitted out by my Friends
in the most liberal manner.—Join my Ship.—Delighted with my new Situation.
—Account of my Messmates, and other Matters.

I had continued some months in this course of life, and was

frequently reduced to very great shifts; on these occasions I had
recourse to those accommodating persons, called pawnbrokers, who
obligingly lend money at the moderate interest of seventy-three
hundred per cent.! as has been clearly proved in a late publication. I
sometimes raised the wind by my old expedient of obtaining goods
on credit, called in the cant language maceing: these I converted
into ready money for immediate purposes. By such artifices I
contrived to support a genteel appearance, though sometimes
bordering on the shabby. My principal enjoyments, indeed, were not
of the most extravagant nature, with the exception of theatrical
amusements. I commonly spent my evenings (if not at the Blue
Lion), at some genteel porter or chop-house, frequented chiefly by
attornies’ clerks and the neighbouring tradesmen; where I enjoyed a
pipe and an evening paper, and at the same time listened to the
conversation of the surrounding guests, amusing myself with their
various characters and peculiarities. I frequently dined, from the
same motives, at different ordinaries, particularly one at the
Saracen’s-Head, Snow-Hill.
One day, after dining at this house, I adjourned to the coffee-
room, and, seating myself in a box, called for a glass of negus, and
a daily paper. I was soon joined by a gentleman of a very
prepossessing aspect, who I perceived, by the buttons on his coat,
to be a warrant officer in the navy. After the customary compliments,
we entered into conversation; and he soon let me understand that
he was surgeon of a frigate called the Astræa.
Delighted with an opportunity of expatiating on my favourite topic,
I asked many questions concerning the service; and the eagerness
of my manner convinced this gentleman that I had a strong
inclination to enter into it. He appeared pleased with my vivacity of
manner, and the pertinence of my remarks; and having extracted
from me a confession of my inclinations, and a brief account of my
family and expectancies, he, with the greatest kindness, undertook
to write in my favour to Captain Dacres, his commander, who was
then at his seat in Devonshire, the frigate being in dock at
Sheerness; assuring me there was little doubt of my obtaining a
midshipman’s birth, provided my friends would consent to it, and fit
me out in a suitable manner for the occasion. I was in raptures at
the result of this fortunate meeting; and expressing the warmest
gratitude for such friendship shewn to an utter stranger, assured Mr.
Kennedy (for this was the surgeon’s name), of my choice being
approved by my grandfather, who anxiously wished me to fix on
some profession, which might afford a prospect of future
independence. It was therefore settled that Mr. Kennedy should
write to the Captain without delay, and that I should immediately
entreat my grandfather to sanction my views, and to write also to
Capt. Dacres, requesting his approbation and commands respecting
me. Mr. Kennedy further recommended that I should, in the interim,
take a trip to Sheerness, and wait on the commanding-officer, Mr.
Rowley, who was then, with the Astræa’s crew, on board the
Carolina hulk, during the frigate’s repair. For this end he furnished
me with a line of introduction to Lieut. Rowley, and added that he
should himself be at Sheerness, where his family resided, in a few
days. He observed that I should be much amused with the
excursion, as I had never been on board a king’s ship, and that I
should also open an acquaintance with the young gentlemen who, in
all probability, were to be my future messmates and companions.
I immediately wrote in the most pressing terms to my grandfather,
assuring him that his refusal to ratify my choice would render me
completely miserable; but that if I was indulged in my desire, I
would never again become troublesome to any of my friends, or
betray any future signs of inconstancy or caprice. I concluded with
expressing my hopes of his receiving a favourable answer from the
captain, and that I should in a few days have this intimation
communicated in a letter from himself, approving of the steps I had
taken.
Having so done, I equipped myself as handsomely as my means
would admit, and the next day set out for Sheerness, where I
arrived in high spirits, and proceeded on board the Carolina. On
presenting my credentials to the first lieutenant, I was very politely
received, and invited to the ward-room, where I was introduced to
several other officers, who, on learning my intentions, and that I was
recommended by the doctor, welcomed me in the kindest terms, and
highly commended my spirit. After refreshing myself, Mr. Rowley
sent for a midshipman, whom he requested to shew me every
attention, and to introduce me to his messmates as a person likely
soon to become a brother Mid. This young man obeyed with the
greatest pleasure, and I soon became perfectly at home. My
conductor next took me through every part of the hulk, and I was
both pleased and astonished at the novelty of the objects I met
with. The hulk had been a line-of-battle ship, and her interior was, of
course, extensive and capacious. The decks were crowded with
hucksters, who exposed provisions, fruits, and other articles for sale.
Groups of sailors with their girls were seen dancing to the music of a
fiddle or a fife; others were engaged at cards, draughts, &c. Grog
was every where the order of the day; but still the most perfect
regularity and cleanliness pervaded every part of this wooden world.
Having satisfied my curiosity, I returned to the midshipmen’s birth,
which was merely a large space in the after-part of the orlop deck,
enclosed with canvass, to render it warm and private. I obtained
from these young gentlemen a list of such clothes, books,
instruments, &c., as were requisite to form a genteel out-fit; and an
estimate of the probable cost of the whole, which I found to be at
least 80l. I now received, by the ward-room steward, an invitation to
dine with the lieutenants, &c. The party was increased by the
company of some ladies from the shore, and the repast was
excellent. Wit, wine, and grog, abounded, and I now conceived I
had found the sphere of life in which I could enjoy unalloyed
happiness. After sitting a reasonable time, I expressed a wish to
view the town of Sheerness, the dock-yard, &c., and requested Mr.
Rowley to permit the midshipman before-mentioned to go on shore
with me. To this he kindly assented, and ordered a boat to be
manned on purpose; stipulating, however, that we should return at a
reasonable hour, and that I would make use of a spare cot, in the
cabin of the third lieutenant, who was absent on leave. My kind
young friend shewed me the objects most worthy of remark; and we
then adjourned to the Marlborough-head inn, where we took some
coffee, and about eight o’clock returned to the hulk. My conductor,
having reported his return to the first lieutenant, wished me good
night, and I took a seat at the ward-room table, where the party was
now reduced to the first and second lieutenants, the master, purser,
and lieutenant of marines. I now found that grog was to be the
order of the night also; but as I had never been accustomed to that
beverage, in the way they drank it, I could not help betraying my
dislike; on which Mr. Rowley, in a good-humoured way, assured me
that I should never make a sailor without I could learn to drink grog.
I was therefore constrained to make a glass, which I did pretty stiff,
as the sailors term it; and its inspiring qualities soon caused me to
banish all reserve. Mr. Rowley conversed with great appearance of
regard on my future prospects; gave me a most amiable character of
the captain; and instructed me in the outlines of my duty, as well as
in the conduct I ought to pursue for my own advantage. He
informed me, the Astræa would be undocked in about three weeks,
and that I had therefore no time to lose in equipping myself.
After as pleasant and rational an evening as I ever remember to
have spent, I retired to rest in the neat little cabin, I have before
mentioned; and slept for the first time in my life on salt-water. The
next morning, after breakfast, I had the pleasure of seeing my
worthy friend, the surgeon, who had just arrived from town. He
insisted on my dining with himself and family ashore; I therefore
took a respectful leave of Mr. Rowley, and the other gentlemen in the
ward-room; a most cordial farewell of my brother Mids, as I was
proud to call them; and, receiving the wishes of all parties for my
speedy return to join the ship, I accompanied Mr. Kennedy on shore.
I spent a pleasant day at his lodgings, and in the evening set out on
my return to London, highly pleased with my excursion, and
indulging the most pleasing hopes of future happiness.
In a week’s time, I had the pleasure of receiving a letter from my
grandfather, wherein he informed me that my entreaties were
granted, though with much reluctance on the part of most of my
friends; and enclosing a handsome letter from Captain Dacres, who
expressed his willingness to receive me as a midshipman on board
His Majesty’s ship Astræa, out of respect to the recommendation of
his surgeon, and because he would not defeat the hopes of a young
man who might one day prove an ornament to the service. My
grandfather concluded with desiring I would immediately come down
to S⸺shire, for the purpose of taking leave of my friends and
receiving the necessary sum to defray the expenses of my
equipment.
I obeyed this welcome summons without delay, and, after a stay
of several days at S⸺, I took an affectionate leave of all my
relatives, who could not divest themselves of anxiety on my account;
and returned to town with nearly 100l. in my pocket, my other
friends having contributed liberally to assist my grandfather in this
emergency.
On arriving in town, I took lodgings in the New Exchange Coffee-
house in the Strand, being a house frequented by naval officers, and
to which Lieutenant Rowley had recommended me. My first care was
to purchase a large and strong chest, which having sent to my
lodgings, I proceeded to buy linen, bedding, uniforms, books, &c.
&c.; and of course, did not forget an elegant hanger, a dirk, and a
cocked-hat, richly bound with gold lace.
 I was so diligent in this affair, that in ten days I had every thing
ready; and, embarking with my luggage on board a Sheerness
packet, the same day sailed down the river Thames. After a
favourable passage, we arrived at Sheerness; and, as we passed
through the fleet, I had the pleasure of seeing my ship lashed
alongside the Carolina, having the preceding day come out of dock.
As soon as the sloop anchored, I went on board the hulk, and was
received with kind encouragement by my superior officers, and with
the most lively pleasure by my future messmates. Mr. Rowley very
kindly sent the cutter to bring my chest, &c., from the packet; and I
was then formally introduced by that gentleman to the midshipmen’s
birth. After a jovial evening, spent in festivity and merriment, I
retired to my hammock, in which I slept well, notwithstanding the
novelty of my situation. The next day was spent in overhauling my
chest, as the young tars termed an inspection of its contents; and I
had the pleasure to find, that my judgment and taste in the
purchases I had made, were universally approved of. Day after day
increased the happiness I felt in my novel and respectable situation;
and my gratitude to those who were, under Providence, the
promoters of my good fortune.
The mess of which I was become a member, consisted of eleven
persons; namely, nine midshipmen of different ages, the captain’s
clerk, and surgeon’s mate: the latter was a most curious character, a
Welshman named Jones; and reminded me, on a very short
acquaintance, of Morgan in Roderic Random, whom he resembled in
many particulars: and I soon found that he was, like him, a kind of
butt for the others to exercise their waggery upon; but in the main
he was a good-hearted, inoffensive young man. The captain’s clerk
was a genteel youth, who had served under his present commander
several years, and was in expectation of being soon promoted to the
rank of purser, through his interest. Of the rest, two or three were
midshipmen who had also served nearly the limited term; one a
master’s mate, and the rest youngsters of different ages. The whole
were of good families, and liberally supported by their friends. I was
called upon to subscribe my monthly quota towards the mess, which