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Methods in
Molecular Biology 1751

Yejun Wang
Ming-an Sun Editors

Transcriptome
Data Analysis
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
Transcriptome Data Analysis

Methods and Protocols

Edited by

Yejun Wang
Department of Cell Biology and Genetics, School of Basic Medicine, Shenzhen University
Health Science Center, Shenzhen, China

Ming-an Sun
Epigenomics and Computational Biology Lab, Biocomplexity Institute of Virginia Tech,
Blacksburg, VA, USA
Editors
Yejun Wang Ming-an Sun
Department of Cell Biology and Epigenomics and Computational Biology Lab
Genetics, School of Basic Medicine Biocomplexity Institute of Virginia Tech
Shenzhen University Health Blacksburg, VA, USA
Science Center
Shenzhen, China

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7709-3 ISBN 978-1-4939-7710-9 (eBook)
https://doi.org/10.1007/978-1-4939-7710-9
Library of Congress Control Number: 2018933577

© Springer Science+Business Media, LLC 2018

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Preface

As sequencing technology improves and costs decrease, more and more laboratories are
performing RNA-Seq to explore the molecular mechanisms of various biological pheno-
types. Due to the increased sequencing depth available, the purposes of transcriptome
studies have also been expanded extensively. In addition to the conventional uses for gene
annotation, profiling, and expression comparison, transcriptome studies have been applied
for multiple other purposes, including but not limited to gene structure analysis, identifica-
tion of new genes or regulatory RNAs, RNA editing analysis, co-expression or regulatory
network analysis, biomarker discovery, development-associated imprinting studies, single-
cell RNA sequencing studies, and pathogen–host dual RNA sequencing studies.
The aim of this book is to give comprehensive practical guidance on transcriptome data
analysis with different scientific purposes. It is organized in three parts. In Part I, Chapters 1
and 2 introduce step-by-step protocols for RNA-Seq and microarray data analysis, respec-
tively. Chapter 3 focuses on downstream pathway and network analysis on the differentially
expressed genes identified from expression profiling data. Unlike most of the other proto-
cols, which were command line-based, Chapter 4 describes a visualizing method for tran-
scriptome data analysis. Chapters 5–11 in Part II give practical protocols for gene
characterization analysis with RNA-Seq data, including alternative spliced isoform analysis
(Chapter 5), transcript structure analysis (Chapter 6), RNA editing (Chapter 7), and
identification and downstream data analysis of microRNA (Chapters 8 and 9), lincRNA
(Chapter 10), and transposable elements (Chapter 11). In Part III, protocols on several new
applications of transcriptome studies are described: RNA–protein interactions (Chapter 12),
expression noise analysis (Chapter 13), epigenetic imprinting (Chapter 14), single-cell RNA
sequencing applications (Chapter 15), and deconvolution of heterogeneous cells
(Chapter 16). Some chapters cover more than one application. For example, Chapter 5
also presents the analysis of single molecule sequencing data in addition to alternative
splicing analysis; Chapter 12 also gives solutions for the analysis of small RNAs in bacteria.
Some topics were not included in this volume due to various factors, e.g., analysis on circular
RNAs, metatranscriptomics, biomarker identification, and dual RNA-Seq. For circular
RNAs, there are numerous published papers or books with protocols that can be followed.
Metatranscriptomics is a new technique and data-oriented methods for analysis are still
lacking. For most other applications, the core protocols for data processing and analysis are
the same as presented in the chapters of this volume.

Shenzhen, China Yejun Wang

Blacksburg, VA, USA Ming-an Sun

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I GENERAL PROTOCOLS ON TRANSCRIPTOME DATA ANALYSIS

1 Comparison of Gene Expression Profiles in Nonmodel
Eukaryotic Organisms with RNA-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Han Cheng, Yejun Wang, and Ming-an Sun
2 Microarray Data Analysis for Transcriptome Profiling. . . . . . . . . . . . . . . . . . . . . . . . 17
Ming-an Sun, Xiaojian Shao, and Yejun Wang
3 Pathway and Network Analysis of Differentially Expressed
Genes in Transcriptomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Qianli Huang, Ming-an Sun, and Ping Yan
4 QuickRNASeq: Guide for Pipeline Implementation
and for Interactive Results Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Wen He, Shanrong Zhao, Chi Zhang, Michael S. Vincent,
and Baohong Zhang

PART II OBJECTIVE-SPECIALIZED TRANSCRIPTOME DATA ANALYSIS

5 Tracking Alternatively Spliced Isoforms from Long Reads

by SpliceHunter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Zheng Kuang and Stefan Canzar
6 RNA-Seq-Based Transcript Structure Analysis with TrBorderExt . . . . . . . . . . . . . 89
Yejun Wang, Ming-an Sun, and Aaron P. White
7 Analysis of RNA Editing Sites from RNA-Seq Data Using GIREMI. . . . . . . . . . . 101
Qing Zhang
8 Bioinformatic Analysis of MicroRNA Sequencing Data . . . . . . . . . . . . . . . . . . . . . . 109
Xiaonan Fu and Daoyuan Dong
9 Microarray-Based MicroRNA Expression Data Analysis
with Bioconductor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Emilio Mastriani, Rihong Zhai, and Songling Zhu
10 Identification and Expression Analysis of Long Intergenic
Noncoding RNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Ming-an Sun, Rihong Zhai, Qing Zhang, and Yejun Wang
11 Analysis of RNA-Seq Data Using TEtranscripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Ying Jin and Molly Hammell

vii
viii Contents

PART III NEW APPLICATIONS OF TRANSCRIPTOME

12 Computational Analysis of RNA–Protein Interactions

via Deep Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Lei Li, Konrad U. Förstner, and Yanjie Chao
13 Predicting Gene Expression Noise from Gene Expression Variations . . . . . . . . . . 183
Xiaojian Shao and Ming-an Sun
14 A Protocol for Epigenetic Imprinting Analysis with RNA-Seq Data . . . . . . . . . . . 199
Jinfeng Zou, Daoquan Xiang, Raju Datla, and Edwin Wang
15 Single-Cell Transcriptome Analysis Using SINCERA Pipeline . . . . . . . . . . . . . . . . 209
Minzhe Guo and Yan Xu
16 Mathematical Modeling and Deconvolution of Molecular
Heterogeneity Identifies Novel Subpopulations in Complex Tissues. . . . . . . . . . . 223
Niya Wang, Lulu Chen, and Yue Wang

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Contributors

STEFAN CANZAR  Gene Center, Ludwig-Maximilians-Universit€ a t München,

Munich, Germany
YANJIE CHAO  Institute of Molecular Infection Biology, University of Würzburg,
Würzburg, Germany; Department of Molecular Biology and Microbiology, Howard Hughes
Medical Institute, Tufts University School of Medicine, Boston, MA, USA
LULU CHEN  Department of Electrical and Computer Engineering, Virginia Polytechnic
Institute and State University, Arlington, VA, USA
HAN CHENG  Key Laboratory of Rubber Biology, Ministry of Agriculture, Rubber Research
Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, P.R.
China
RAJU DATLA  National Research Council Canada, Saskatoon, SK, Canada
DAOYUAN DONG  Department of Chemistry and Biochemistry, University of the Sciences,
Philadelphia, PA, USA
KONRAD U. FÖRSTNER  Institute of Molecular Infection Biology, University of Würzburg,
Würzburg, Germany
XIAONAN FU  Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA
MINZHE GUO  The Perinatal Institute, Section of Neonatology, Perinatal and Pulmonary
Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
MOLLY HAMMELL  Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
WEN HE  Early Clinical Development, Pfizer Worldwide R&D, Cambridge, MA, USA
QIANLI HUANG  School of Biological and Medical Engineering, Hefei University of
Technology, Hefei, China
YING JIN  Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
ZHENG KUANG  Department of Immunology, The University of Texas Southwestern Medical
Center, Dallas, Texas, USA
LEI LI  Institute of Molecular Infection Biology, University of Würzburg,
Würzburg, Germany; Division of Biostatistics, Dan L. Duncan Cancer Center, Baylor
College of Medicine, Houston, TX, USA
EMILIO MASTRIANI  Systemomics Center, College of Pharmacy, Harbin Medical University,
Harbin, China; Genomics Research Center (State-Province Key Laboratories of
Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, China
XIAOJIAN SHAO  Department of Human Genetics, McGill University, Montréal, Canada;
The McGill University and Génome Québec Innovation Centre, Montréal, QC, Canada
MING-AN SUN  Epigenomics and Computational Biology Lab, Biocomplexity Institute of
Virginia Tech, Blacksburg, VA, USA
MICHAEL S. VINCENT  Inflammation and Immunology Research Unit, Pfizer Worldwide
R&D, Cambridge, MA, USA
EDWIN WANG  Department of Experimental Medicine, McGill University,
Montreal, QC, Canada; Center for Bioinformatics, McGill University,
Montreal, QC, Canada; Center for Health Genomics and Informatics, University of
Calgary Cumming School of Medicine, Calgary, AB, Canada; Department of Biochemistry
and Molecular Biology, University of Calgary Cumming School of Medicine,
Calgary, AB, Canada; Department of Medical Genetics, University of Calgary Cumming

ix
x Contributors

School of Medicine, Calgary, AB, Canada; Department of Oncology, University of Calgary

Cumming School of Medicine, Calgary, AB, Canada; Alberta Children’s Hospital
Research Institute, Calgary, AB, Canada; Arnie Charbonneau Cancer Research Institute,
Calgary, AB, Canada; O’Brien Institute for Public Health, Calgary, AB, Canada; Wang
Lab, Health Science Centre, University of Calgary, Calgary, AB, Canada
NIYA WANG  Department of Electrical and Computer Engineering, Virginia Polytechnic
Institute and State University, Arlington, VA, USA
YEJUN WANG  Department of Cell Biology and Genetics, School of Basic Medicine, Shenzhen
University Health Science Center, Shenzhen, PR China
YUE WANG  Department of Electrical and Computer Engineering, Virginia Polytechnic
Institute and State University, Arlington, VA, USA
AARON P. WHITE  Vaccine and Infectious Disease Organization, University of Saskatchewan,
Saskatoon, SK, Canada
DAOQUAN XIANG  National Research Council Canada, Saskatoon, SK, Canada
YAN XU  The Perinatal Institute, Section of Neonatology, Perinatal and Pulmonary Biology,
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Division of
Biomedical Informatics, Cincinnati Children’s Hospital Medical Center,
Cincinnati, OH, USA
PING YAN  School of Biological and Medical Engineering, Hefei University of Technology,
Hefei, China
RIHONG ZHAI  School of Public Health, Shenzhen University Health Science Center,
Shenzhen, China
BAOHONG ZHANG  Early Clinical Development, Pfizer Worldwide R&D,
Cambridge, MA, USA
CHI ZHANG  Early Clinical Development, Pfizer Worldwide R&D, Cambridge, MA, USA
QING ZHANG  Integrative Biology and Physiology, The University of California, Los Angeles
(UCLA), Los Angeles, CA, USA
SHANRONG ZHAO  Early Clinical Development, Pfizer Worldwide R&D,
Cambridge, MA, USA
SONGLING ZHU  Systemomics Center, College of Pharmacy, Harbin Medical University,
Harbin, China; Genomics Research Center (State-Province Key Laboratories of
Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, China
JINFENG ZOU  National Research Council Canada, Montreal, QC, Canada
 Part I

General Protocols on Transcriptome Data Analysis

 Chapter 1

Comparison of Gene Expression Profiles in Nonmodel

Eukaryotic Organisms with RNA-Seq
Han Cheng, Yejun Wang, and Ming-an Sun

Abstract
With recent advances of next-generation sequencing technology, RNA-Sequencing (RNA-Seq) has
emerged as a powerful approach for the transcriptomic profiling. RNA-Seq has been used in almost every
field of biological studies, and has greatly extended our view of transcriptomic complexity in different
species. In particular, for nonmodel organisms which are usually without high-quality reference genomes,
the de novo transcriptome assembly from RNA-Seq data provides a solution for their comparative tran-
scriptomic study. In this chapter, we focus on the comparative transcriptomic analysis of nonmodel
organisms. Two analysis strategies (without or with reference genome) are described step-by-step, with
the differentially expressed genes explored.

Key words Nonmodel organism, RNA-Seq, Next-generation sequencing, Differential expression,

Transcriptome, de novo transcriptome assembly

1 Introduction

Recent advantages in next-generation sequencing have enabled the

development of RNA-Seq—a powerful approach allowing the
investigation of transcriptome at unsurpassed resolution [1].
RNA-Seq has the potential to reveal unprecedented complexity of
the transcriptomes, to provide quick insights into the gene struc-
ture without the requirement of reference genome, to expand the
identification for the genes of interest, to develop functional molec-
ular markers, to quantify gene expression, and to compare gene
expression profiles [2]. These advantages have made RNA-Seq the
most popular method for transcriptome analysis [3]. In particular,
unlike microarray which is another popular method for transcrip-
tome profiling but needs to be designed according to presequenced
reference genome, RNA-Seq could be applied for the transcrip-
tomic study in nonmodel organisms [4]. Next-generation sequenc-
ing becomes more affordable in recent years, making RNA-Seq
more and more popular in ordinary molecular biology laboratory.

Yejun Wang and Ming-an Sun (eds.), Transcriptome Data Analysis: Methods and Protocols, Methods in Molecular Biology,
vol. 1751, https://doi.org/10.1007/978-1-4939-7710-9_1, © Springer Science+Business Media, LLC 2018

3
4 Han Cheng et al.

RNA-Seq has already been used in almost every field of biological

studies, and has greatly extended our view of transcriptomic com-
plexity in different species. However, the huge amounts of reads
generated by RNA-Seq pose great challenges to the assembly and
analysis of complete transcriptomes. Fortunately, recent progresses
in bioinformatics provided powerful tools for RNA-Seq analysis of
species lacking high-quality reference genome.
In nonmodel organisms, de novo transcriptome assembly is the
first step for constructing a reference when the complete genome
sequences are absent. In recent years, several tools have been devel-
oped for de novo transcriptome assembly, such as Trinity,
SOAPdenovo-Trans, and ABYSS [4–6]. These tools each have
their own merits for dealing with different types of genomes. The
short reads are then mapped to the reference transcriptome, and
the read counts of each transcript are normalized and compared
between each sample. In this step, we usually use RSEM for quan-
tifying transcript abundances [7]. The final step is to annotate each
transcript and to visualize the expression results.
The tools mentioned above greatly facilitate transcriptome
assembly and promote RNA-Seq studies in the nonmodel organ-
isms. In recent years, a great number of studies appeared to identify
differentially expressed (DE) genes between specific treatments or
tissues [8–13]. In this chapter, we give a step-by-step protocol to
assemble a reference transcriptome and to explore DE genes from
RNA-Seq data.

2 Materials

2.1 Software All the software packages need to be installed in your workstation in
Packages advance. Because most bioinformatics tools are designed for Linux
operating systems, here we demonstrate each step according to 64-bit
Ubuntu OS. For the convenience of running the commands in
your working directory, add the folders containing your executes
into your PATH environment variable so that the executes could be
used directly when you type their names. To be noted, some software
used in this protocol may be not the latest version. In such case, it is
highly encouraged to download the latest version for use.

2.1.1 SRA Toolkit Download the SRA toolkit [14], unpack the tarball to your desti-
nation directory (e.g., /home/your_home/soft/), and add the
executables path to your PATH, type:
wget http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/current/sratool
kit.current-centos_linux64.tar.gz.
 Non-Model Organisms Transcriptome Analysis 5

tar xzf –C /home/your_home/soft/ sratoolkit.current- centos_

linux64.tar.gz

export PATH¼/home/your_home/soft/sratoolkit.2.7.0-
ubuntu64/bin:$PATH

2.1.2 FastQC Download the FastQC package [15], unpack and add the directory
to your PATH.
wget http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
fastqc_v0.10.1.zip

unzip fastqc_v0.10.1.zip –d /home/your_home/soft/

export PATH¼/home/your_home/soft/FastQC:$PATH

2.1.3 Trinity Download the Trinity package [4], unpack, and add the directory
to your PATH.
wget https://github.com/trinityrnaseq/trinityrnaseq/archive/
v2.2.0.tar.gz.

tar xzf –C /home/your_home/soft/ trinityrnaseq-2.2.0.tar.gz

export PATH¼/home/your_home/soft/trinityrnaseq-2.2.0:
$PATH

export PATH¼/home/your_home/soft/trinityrnaseq-2.2.0/
util:$PATH

2.1.4 RSEM Download the RSEM package [7], unpack, and add the RSEM
directory to your PATH.
wget https://github.com/deweylab/RSEM/archive/v1.2.8.tar.gz

tar xzf –C /home/your_home/soft/ RSEM-1.2.8.tar.gz

export PATH¼/home/your_home/soft/rsem-1.2.8:$PATH

2.1.5 R Download R [16], unpack and then install.

wget https://cran.r-project.org/src/base/R-3/R-3.2.2.tar.gz

tar zxf –C /home/your_home/soft/ R-3.2.2.tar.gz

cd /home/your_home/soft/R-3.2.2

./configure ./configure --prefix¼/home/your_home/bin

6 Han Cheng et al.

make

make check

make install

2.1.6 Bowtie2 Download Bowtie2 package [17], unpack, and then add Bowtie2
directory to your PATH.
wget http://jaist.dl.sourceforge.net/project/bowtie-bio/bow
tie2/2.2.6/bowtie2-2.2.6-linux-x86_64.zip

unzip bowtie2-2.2.6-linux-x86_64.zip -d /home/your_home/

soft/

export PATH¼/home/your_home/soft/ bowtie2-2.2.6:

$PATH

2.1.7 Tophat Download Tophat [18], unpack and install, and then add the
(See Note 1) directory to your PATH.
wget http://ccb.jhu.edu/software/tophat/downloads/tophat-
2.0.9.Linux_x86_64.tar.gz

tar zxf tophat-2.0.9.Linux_x86_64.tar.gz

cd tophat-2.0.9.linux_x86_64

./configure --prefix¼/home/your_home/soft/tophat2

make

make install

export PATH¼/home/your_home/soft/tophat2:$PATH

2.1.8 Cufflinks Download Cufflinks [19], unpack and then add the directory to
your PATH.
wget http://cole-trapnell-lab.github.io/cufflinks/assets/down
loads/cufflinks-2.2.1.Linux_x86_64.tar.gz

tar xzf –C /home/your_home/soft/ cufflinks-2.2.1.Linux_x86_

64.tar.gz
 Non-Model Organisms Transcriptome Analysis 7

export PATH¼/home/your_home/soft/cufflinks-2.2.1.Linux_
x86_64:$PATH

2.1.9 EBSeq EBSeq [20] is an R Bioconductor package for gene and isoform
differential expression analysis of RNA-Seq data. For installation,
just start R and enter:
source("https://bioconductor.org/biocLite.R")

biocLite("EBSeq")

2.1.10 DESeq DESeq [21] is an R Bioconductor package for differential expres-

sion analysis with reads count data. To install it, start R and enter:
source("https://bioconductor.org/biocLite.R")

biocLite("DESeq")

2.2 Data Samples Most public RNA-Seq data could be downloaded from NCBI SRA
database (https://www.ncbi.nlm.nih.gov/sra) (see Note 2). In this
protocol, we use RNA-Seq data set from the rubber tree. This data set
includes six samples from control and cold stressed conditions with
three biological replicates, which are denoted as “control” and “cold.”

3 Methods

Download the RNA-Seq data from NCBI SRA database and place
the files in your working directory (e.g., /home/your_name/
NGS/SRA). Run the commands as demonstrated in this protocol
in your working directory (see Notes 3 and 4).

3.1 RNA-Seq Data 1. Generate FASTQ files from SRA files. To extract FASTQ files
Quality Control from downloaded sra files, and put them in a new folder “fq”,
go to your NGS data directory and type (see Note 5):
fastq-dump -O ./fq --split-files ./SRA/SRR*.sra
2. Quality controlling by fastQC (see Note 6).
fastqc -o ./qc -f fastq ./fq/Sample*.fastq
3. Remove reads of low quality (optional). In most cases, the low
quality reads have been removed when the sequences were
transferred from the service supplier. In this example, the
FASTQ file has been filtered when submitted to the NCBI
SRA database (see Note 7).
fastq_quality_filter -Q33 -v -q 30 -p 90 -i fq/Sample*.fastq
-o fq/Sample*.fastq
8 Han Cheng et al.

3.2 Gene Expression In most cases, nonmodel organisms do not have reference genome.
Analysis Without We therefore use no reference genome analysis strategy to compare
Reference Genome gene expression profiles and to find DE genes. This strategy first
assembles a reference transcriptome from the RNA-Seq data, and
then maps the reads to the reference transcriptome and calculates
gene expression. In this protocol, we use Trinity to assemble transcrip-
tome, and then use RSEM to calculate reads counts, finally utilize two
popular packages, EBSeq and DESeq, to find DE genes respectively.
1. Reference transcriptome assembly. The Trinity program [4]
can assemble the reads in all the sample files into one reference
transcriptome. Then the reference transcriptome can be used
for gene expression analysis. For paired-end RNA-Seq with
read1 (*_1.fastq) and read2 (*_2.fastq), the reference tran-
scriptome could be assembled by typing:
Trinity.pl --JM 500G --seqType fq --left fq/Sample*_1.fastq
--right fq/Sample*_2.fastq --output trinity_out --min_
kmer_cov 5 --CPU 32

(see Note 8)
Trouble shooting: In some cases, the Trinity program will
stop due to short of memory when executing the “butterfly_
commands”. You may go to the results directory trinity_out/
chrysalis/ and check if the “butterfly_commands” file exists.
Then use the following commands to continue the assembly.
cmd_process_forker.pl -c trinity_out/chrysalis/butterfly_
commands --CPU 10 --shuffle;

find trinity_out/chrysalis -name "*allProbPaths.fasta" -exec

cat {} \; > trinity_out/Trinity.fasta;
You will find a “Trinity.fasta” file in the output directory, which
is the assembled reference transcriptome of all the reads. You
can also check the reference transcriptome statistics by running
the TrinityStats.pl script provided by Trinity package:
TrinityStats.pl trinity_out/Trinity.fasta
2. Gene expression quantification with RSEM. RSEM is an accu-
rate and user-friendly tool for quantifying transcript abun-
dances from RNA-Seq data and it does not rely on the
existence of a reference genome [7]. Therefore, it is particularly
useful for expression quantification with de novo transcriptome
assemblies. The RSEM program includes just two scripts (rsem-
prepare-reference and rsem-calculate-expression), which invokes
 Non-Model Organisms Transcriptome Analysis 9

Bowtie [22] for read alignment. The first step is to extract and
preprocess the reference sequences and then builds Bowtie
indices.
mkdir rsem

cd rsem

mkdir tmp

extract-transcript-to-gene-map-from-trinity ../trinity_out/
Trinity.fasta tmp/unigenes.togenes

rsem-prepare-reference --transcript-to-gene-map tmp/

unigenes.togene ../trinity_out/Trinity.fasta tmp/unigenes

Then the RNA-Seq reads in each sample are aligned to the

Bowtie indices and their relative abundances are calculated. The
tasks are handled by the rsem-calculate-expression script. By default,
RSEM uses the Bowtie alignment program to align reads, with
parameters specifically chosen for RNA-Seq quantification. The
rsem-calculate-expression script processes the reads in each sample.
A short Bash script will be much easier to handle large amount of
samples in one analysis.
export k

for ((k¼1;k<6;kþ¼1));do

rsem-calculate-expression -p 24 --bowtie-chunkmbs 512

--paired-end --no-bam-output --forward-prob 0.0 fq/Sample
${k}_1.fq fq/Sample${k}_2.fq tmp/unigenes rsem/Sample${k};

done

The rsem-calculate-expression script produces two files with “.

results” suffix, in which the “.gene.results” file calculate TPM and
FPKM for each gene, whereas the “.transcripts.results” listed the
TPM and FPKM for each transcript. The file structures are as follow:
The “Sample.genes.results” file:
gene_id transcript_id(s) length effective_length expected_count TPM FPKM

c0.graph_c0 c0.graph_c0_seq1 745.00 690.31 14.00 2.43 1.79

c1.graph_c0 c1.graph_c0_seq1 262.00 207.46 1.00 0.58 0.43

10 Han Cheng et al.

The “Sample.transcripts.results” file:

transcript_id gene_id length effective_length expected_count TPM FPKM IsoPct

c0.graph_c0_seq1 c0.graph_c0 745 690.31 14.00 2.43 1.79 100.00

c1.graph_c0_seq1 c1.graph_c0 262 207.46 1.00 0.58 0.43 100.00

3. Differentially expressed gene identification with EBSeq. EBSeq
is an R package for exploring DE genes and isoforms from
RNA-Seq data, which is based on empirical Bayesian method
and aims to identify DE isoforms between two or more
biological samples [20]. EBSeq processes counts matrix files
generated by RSEM, and calculates the expression of each gene
in each sample.
RSEM provides several wrappers which could invoke EBSeq to
identify differentially expressed genes. This is the easier way to use
EBSeq. Merge each single counts file to generate a matrix file with
the following commands:
rsem-generate-ngvector ../trinity_out/Trinity.fasta cov5_trinity

rsem-generate-data-matrix Sample*.genes.results >

genes.counts.matrix

Then use the following commands to obtain DE genes:

rsem-run-ebseq --ngvector cov5_trinity.ngvec genes.
counts.matrix 3,3 GeneMat.results

rsem-control-fdr GeneMat.results 0.05 GeneMat.de.txt

(see Note 9)
Alternatively, you can also use EBSeq in a native way for DE
gene identification. In R console, type:
library(“EBSeq”)

setwd("/path/to/your/directory/rsem/")

GeneMat <- data.matrix(read.table(file¼"genes.counts.

matrix"))

NgVec <- scan(file¼"cov5_trinity.ngvec", what¼0, sep¼"\n")

 Non-Model Organisms Transcriptome Analysis 11

Condition ¼ factor(c("Control","Control","Control","Cold",
"Cold","Cold"))

GeneSizes ¼ MedianNorm(GeneMat)

GeneEBOut ¼ EBTest (Data¼GeneMat, Conditions¼Condi-

tion,sizeFactors¼GeneSizes, maxround¼10)

GeneEBDERes¼GetDEResults(GeneEBOut, FDR¼0.05)

(see Note 9)
For more detailed function introduction, please refer EBSeq
vignette [20].
4. Differentially expressed gene identification with DESeq. Alter-
natively, you can use DESeq for DE gene identification. DESeq
is a R package to analyze sequence counts data from RNA-Seq
and test for differential expression [21]. DESeq accepts RSEM
output files for analysis. The first step is to merge each FPKM
count files generated by rsem-calculate-expression script in
RSEM package. The merging step can be performed with
merge_RSEM_frag_counts_single_table.pl scripts from Trinity
package:
TRINITY_HOME/util/RSEM_util/merge_RSEM_frag_
counts_single_table.pl Sample1.genes.results Sample2.genes.results
Sample3.genes.results Sample4.genes.results Sample5.genes.results
>all.genes.counts

Then in R console, type:

library(“DESeq”)

countTable<-read.table("all.genes.counts",header¼T,sep¼
"\t",row.names¼1)

countTable ¼ round(countTable)
(see Note 10)
conditions<-factor(c("Control","Control","Control",
"Cold","Cold","Cold"))

cds<-newCountDataSet(countTable,conditions)

cds<-estimateSizeFactors(cds)

cds<-estimateDispersions(cds)
12 Han Cheng et al.

res <-nbinomTest(cds,"Control","Cold") #call differential

expression

write.table(res, ’compare.csv’,sep¼’\t’,quote¼F,row.names¼F)

head(res)

plotMA(res)

res_sig<-subset(res, padj<0.05);
(see Note 11)
dim(res_sig)

res_sig_order<-res_sig[order(res_sig$padj),]

write.table(res_sig_order, ’difference.txt’,sep¼’\t’,quote¼F,
row.names¼F)
(see Note 12)
For detailed introduction, please refer to DESeq vignette [23].

3.3 Gene Expression Benefiting from genome sequencing projects, many reference gen-
Analysis omes have been published in nonmodel organisms recently. In
with Reference these organisms, the analysis strategy with reference genome can
Genome be adopted. Typically, we first prepare the reference genome files,
then map each reads file to the reference genome, and finally call the
DE genes.
1. Prepare reference genome file. Download the genome files
(sequence fasta file and gff annotation file) from GenBank
database, and then build the bowtie2 index with “bowtie2-
build” command in Bowtie2 package:

bowtie2-build /path/to/genome/HbGenome.fas bowtie-

ref/Hbgenome

(see Note 13)

2. Map reads to reference genome. Map each reads file to the
genome index with tophat2 program, and then assemble tran-
scripts from the reads file with cufflinks program:
tophat2 -o 1th -p 32 -G /path/to/gff/HbGenome.gff3
bowtie-ref/HbGenome /path/to/sample1/Sample1_1.fq/
path/to/sample/Sample1_2.fq
 Non-Model Organisms Transcriptome Analysis 13

cufflinks -p 32 -o 1cl 1th/accepted_hits.bam

You may use a short Bash script to analyze several samples in

one command:
export k;

for ((k¼1;k&lt;6;kþ¼1));do

tophat2 -o ${k}th -p 32 -G /path/to/gff/HbGenome.gff3

bowtie-ref/HbGenome /path/to/sample1/Sample${k}_1.fq/
path/to/sample/Sample${k}_2.fq;

cufflinks -p 32 -o ${k}cl ${k}th/accepted_hits.bam;

done
Then merge all the assembled transcripts files:
ls *cl/transcripts.gtf >assemblies.txt

cuffmerge -p 32 -g /path/to/gff/HbGenome.gff3 -s /
path/to/genome /HbGenome.fas assemblies.txt
(see Note 14)
3. Call differential expression genes with Cuffdiff. Cufflinks
includes a program, “Cuffdiff”, which can be used to find
significant changes in transcript expression, splicing, and pro-
moter use. Cuffdiff requires two types of files: sam (or bam) file
from Tophat program and transcript annotation gtf file from
cufflinks:

cuffdiff -o diff_out/ -b /path/to/genome/Hbgenome.fa

-L Control,Cold -u merged_asm/merged.gtf -p 8 1th/accep-
ted_hits.bam,2th/accepted_hits.bam,3th/accepted_hits.bam
4th/accepted_hits.bam,5th/accepted_hits.bam,6th/accepted_
hits.bam

(see Note 15)

The comparison results will be wrote to “diff_out” directory.
Several comparison results will be found, including cds, isoform,
gene, tss, splicing, and promoter. In most cases, you may be inter-
ested in “gene_exp.diff” file. Then you can extract DE genes from
this file based on your criteria and the adjusted “q_value”. The
content of the diff file:
14 Han Cheng et al.

test_id gene_id gene locus sample_1 sample_2 status value_1 value_2

log2(fold_change) test_stat p_value q_value significant

XLOC_000001 XLOC_000001 - scaffold0001:445549-451760 Control Cold

OK 4.17386 2.62692 -0.668007 -0.799812 0.1381 0.404678 no

4 Notes

1. The Tophat2 was superseded by HISAT2. In this protocol, we

still use old version Tophat for analysis.
2. To simplify the analysis procedure, we use nonmodel Hevea
brasiliensis (rubber tree) RNA-Seq data as the example.
This dataset include two samples (Leaf under control condition,
and cold treated for 24 h), each with three biological replicates.
3. This protocol only shows how to run each analysis steps, and
also gives frequently used options for each command or scripts.
You may also go to check each option of the command and
optimize your own analysis parameters.
4. Please note that the directory structural differences between
this protocol and your own workstation. You should change
the file paths and names according to your own directory.
5. The fastq-dump tool extract reads from SRA package. The
parameter “-O” defines the output directory. “--split-files”
option will enable dumping each read into separate file. Files
will receive suffix corresponding to read number.
6. The results are in the subdirectory under the name of fastq
filename with a “_fastqc” suffix. You may examine the detail
quality check results in “astqc_report.html” file.
7. Add the “-Q33” parameter when meet “fastq_quality_filter:
Invalid quality score value” error.
8. “--JM” option defines how much Giga memory allocated for
the jellyfish to calculate k-mer. --left and --right define the left
and right fastq files for the pair-end seuqencing results. --
min_kmer_cov defines the minimal kmer when calculate the
k-mer number in Inchworm, a high --min_kmer_cov value will
reduce the noise in the assembly and to identify only transcripts
that were relatively highly expressed, but also lose some lowly
expressed transcripts. Define --CPU number for the inchworm
when your server has multiple CPU.
9. This analysis found DE genes at the target FDR of 0.05.
 Non-Model Organisms Transcriptome Analysis 15

10. Expected_counts from RSEM are float numbers because the

reads mapped to multiple locations are assigned to each loca-
tion according to the fractional weighted estimation using an
EM algorithm. However, the DESeq only accepts integer
counts. We therefore use round function to get integer counts.
11. Get DE genes by adjusted p-value less than 0.05.
12. The scripts find DE genes by adjusted p-value less than 0.05,
then export DE gene list to the “difference.txt” file.
13. The bowtie2-build command builds an “Hbgenome” genome
index from genome file “HbGenome.fas”.
14. The program will generate a “merged.gtf” file in “merge-
d_asm” directory.
15. Supply replicate SAMs as comma separated lists for each condi-
tion: Sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.
sam. Separate each condition with space. -L/labels, comma-
separated list of condition labels. Each lable indict one treatment
(condition); The label numbers should equal to conditions.

Acknowledgments

This work is supported by the National Natural Science Foundation

of China (grant No. 31301072).

References
1. Hoeijmakers WAM, Bártfai R, Stunnenberg 8. Chao J, Chen Y, Wu S, Tian W-M (2015)
HG (2013) Transcriptome analysis using Comparative transcriptome analysis of latex
RNA-Seq. Methods Mol Biol 923:221–239 from rubber tree clone CATAS8-79 and
2. Garg R, Jain M (2013) RNA-Seq for transcrip- PR107 reveals new cues for the regulation of
tome analysis in non-model plants. Methods latex regeneration and duration of latex flow.
Mol Biol 1069:43–58 BMC Plant Biol 15:104
3. Wang Z, Gerstein M, Snyder M (2009) 9. Fang Y, Mei H, Zhou B et al (2016) De novo
RNA-Seq: a revolutionary tool for transcrip- Transcriptome analysis reveals distinct Defense
tomics. Nat Rev Genet 10:57–63 mechanisms by young and mature leaves of
4. Grabherr MG, Haas BJ, Yassour M et al (2011) Hevea Brasiliensis (Para rubber tree). Sci Rep
Full-length transcriptome assembly from 6:33151
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Nat Biotechnol 29:644–652 Analysis of stress-responsive gene expression in
5. Xie Y, Wu G, Tang J et al (2014) cultivated and weedy Rice differing in cold
SOAPdenovo-trans: de novo transcriptome stress tolerance. PLoS One 10:e0132100
assembly with short RNA-Seq reads. Bioinfor- 11. Fu J, Miao Y, Shao L et al (2016) De novo
matics 30:1660–1666 transcriptome sequencing and gene expression
6. Simpson JT, Wong K, Jackman SD et al (2009) profiling of Elymus Nutans under cold stress.
ABySS: a parallel assembler for short read BMC Genomics 17:870
sequence data. Genome Res 19:1117–1123 12. Nakashima K, Yamaguchi-Shinozaki K, Shino-
7. Li B, Dewey CN (2011) RSEM: accurate tran- zaki K (2014) The transcriptional regulatory
script quantification from RNA-Seq data with network in the drought response and its cross-
or without a reference genome. BMC Bioin- talk in abiotic stress responses including
formatics 12:323 drought, cold, and heat. Front Plant Sci 5:170
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13. An D, Yang J, Zhang P (2012) Transcriptome 19. Trapnell C, Roberts A, Goff L et al (2012)
profiling of low temperature-treated cassava Differential gene and transcript expression
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tropical plant to cold stress. BMC Genomics and cufflinks. Nat Protoc 7:562–578
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babraham.ac.uk/projects/fastqc/ 21. Anders S, Huber W (2010) Differential expres-
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17. Langmead B, Salzberg SL (2012) Fast gapped- 22. Langmead B, Trapnell C, Pop M, Salzberg SL
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9:357–359 ment of short DNA sequences to the human
18. Kim D, Pertea G, Trapnell C et al (2013) genome. Genome Biol 10:R25
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4:1070
 Chapter 2

Microarray Data Analysis for Transcriptome Profiling

Ming-an Sun, Xiaojian Shao, and Yejun Wang

Abstract
Microarray data have vastly accumulated in the past two decades. Due to the high-throughput characteristic
of microarray techniques, it has transformed biological studies from specific genes to transcriptome level,
and deeply boosted many fields of biological studies. While microarray offers great advantages for expres-
sion profiling, on the other hand it faces a lot challenges for computational analysis. In this chapter, we
demonstrate how to perform standard analysis including data preprocessing, quality assessment, differential
expression analysis, and general downstream analyses.

Key words Microarray, Normalization, Clustering, Differential expression, Bioconductor, Limma,

GeneFilter

1 Introduction

The successful application of microarray for expression analysis

could be traced back to two decades ago [1]. Since then, the
microarray technique has been widely used for expression profiling
in almost every field of biological research [2]. Beyond transcrip-
tion analysis, alternative microarray based techniques have also
been designed for other purposes such as genotyping, DNA
mapping, protein binding, and epigenetic studies [3]. Due to the
high-throughput characteristics of microarray techniques, it has
transformed biological studies from specific genes to transcriptome
level, and deeply boosted many fields of biological studies. Previous
studies showed that microarray is robust for measuring transcrip-
tome [4]. Even though RNA-Seq has emerged in recent years,
microarrays remain popular for measuring gene expression
[5, 6]. In particular, since microarray is cheaper than RNA-Seq, it
has advantages for clinical studies, which may involve a huge
amount of samples. For example, microarray is frequently used in
several comprehensive projects for cancers, including The Cancer
Genome Atlas project [7].

Yejun Wang and Ming-an Sun (eds.), Transcriptome Data Analysis: Methods and Protocols, Methods in Molecular Biology,
vol. 1751, https://doi.org/10.1007/978-1-4939-7710-9_2, © Springer Science+Business Media, LLC 2018

17
18 Ming-an Sun et al.

While microarray offers great advantages for expression

profiling, on the other hand it faces a lot challenges for analysis
[2]. In particular, technical noise could be introduced in microarray
data. Additionally, the challenges of analysis also come from the
tremendous number of probes in microarray, and the few number
of replicates used for most microarray studies. Currently, a large
number of methods have been proposed to deal with problems for
each analysis step, including quality control [8–10], normalization
[11], and differential expression analysis [12–14].
Bioconductor is an open-source, open-development software
project for the analysis and comprehension of high-throughput
data arising from genomics and molecular biology [15]. So far
more than 1000 packages have been released in the Bioconductor.
Importantly, every step for microarray data analysis could find a
solution using packages hosted in Bioconductor project (see Note
1). In this chapter, we show how to implement each step of micro-
array analysis, including quality control, normalization, differential
expression analysis and some general downstream analyses, using
packages mainly from Bioconductor project. In this protocol, data
generated from Affymetrix Mouse Gene 2.0 ST Array (MoGene-
2.0-ST) platform was used for demonstration. However, the analy-
sis procedure described in this protocol could be adjusted for the
analysis of data from other microarray platforms easily.

2 Materials

2.1 Microarray Data This protocol starts with Affymetrix microarray data of CEL format
(see Note 2). The CEL files store the results of the calculated
intensity. In addition to newly generated CEL files in the lab, a
huge amount of published CEL files could be retrieved from several
public resources, in particular ArrayExpress (https://www.ebi.ac.
uk/arrayexpress/) and NCBI Gene Expression Ominibus (GEO;
https://www.ncbi.nlm.nih.gov/geo/). To be noted, ArrayExpress
is specific for microarray data, while GEO also contains other types
of OMICs data.
In this protocol, we use public datasets (GEO accession:
GSE67964) for Affymetrix Mouse Gene 2.0 ST Array (MoGene-
2.0-ST) for demonstration.

2.2 R Packages This protocol involves a number of R packages, thus basic knowl-
edge about R and Bioconductor is essential. The basics of R could
be found from resources such as http://tryr.codeschool.com/. R
and Bioconductor could be installed by following instructions from
http://www.bioconductor.org/install/. Below we briefly summar-
ized the ways for R and Bioconductor packages installation and
loading (see Note 3). For the installation of each package used in
this protocol, it will be described in the corresponding section.
 Microarray Data Analysis 19

R packages could be installed using the install.packages() func-

tion easily. Take ggplot2 package as example, you just need to start
the R console and type:
install.packages("ggplot2")

To install core packages from Bioconductor, type:

source("https://bioconductor.org/biocLite.R")

Then, specific Bioconductor packages could be installed. For

example, to install the oligo package, type:
biocLite("oligo")

After installation, both R or Bioconductor packages could be

loaded by the library() function. Take the oligo package as exam-
ple, to load it, type:
library(oligo)

2.3 Annotation Files Two types of annotation files are required: (1) the probe set anno-
tation, which summarizes the location of all probes on the array, as
well as the probes for each probe set; (2) gene annotation, which
maps the probesets to their corresponding genes.
For most microarray platforms, R Bioconductor packages
providing the annotation information are ready for use (see Note 1).
For example, the two annotation packages for MoGene2.0-ST micro-
array are pd.mogene.2.0.st [16] and mogene20sttranscriptcluster.db
[17], respectively. Since this protocol involves a lot of R Bioconductor
packages, these annotation packages could be incorporated into the
pipeline seamlessly.

3 Methods

3.1 Data We download CEL files from GEO (https://www.ncbi.nlm.nih.

Preprocessing gov/geo/) by searching GEO accession (e.g., GSE67964). This
dataset contains data for wild-type and ROR_alpha_gamma_dKO,
3.1.1 Prepare Data each with four replicates.

3.1.2 Set Work Directory To set the work directory, type:

setwd(“directory_with_CEL_files”)

3.1.3 Read Data into The Bioconductor package “oligo” offers a number of tools for
Memory preprocessing of Affymetrix CEL files, including data import, back-
ground correction, normalization, data summarization and visuali-
zation [18]. In addition, you might need to install and load the
20 Ming-an Sun et al.

probe set annotation package (e.g., pd.mogene.2.0.st for

MoGene2.0-ST platform), if it is failed to be installed automatically
together with “oligo”.
1. To install and load the oligo package, type:
biocLite("oligo")
library(oligo)

2. To get the list of all the CEL files in the directory, type:

cel.files <- list.celfiles()

Or if you only want to read specific CEL files (e.g., celfile1

and celfile2), type:

cel.files <- c(celfile1, celfile2)

3. By default, CEL file names will be specified as sample names.

However, we usually want to respecify sample names, in partic-
ular when the CEL file names are lengthy. The sample names
should be of the same number and order of CEL file names. To
specify sample names manually, type:

sample.names = c("WT1", "WT2", "WT3", "WT4","KO1", "KO2",

"KO3", "KO4")

4. To read CEL files into memory, type:

affy.raw <- read.celfiles(cel.files, sampleNames = sample.

names)

3.1.4 Get Normalized To summarize gene level expression, the probeset annotation for
Gene Expression specific array is required. Take microarray data from mogene.2.0.st
platform as example, the Bioconductor package pd.mogene.2.0.st
[16] is needed.
1. To install and load the annotation library pd.mogene.2.0.st,
type:
biocLite("pd.mogene.2.0.st")
library(pd.mogene.2.0.st)

2. To make reasonable comparison between different samples,

normalization must be performed. Robust Multi-Array Aver-
age (RMA) is the most widely used normalization algorithm.
Meanwhile, there are several other normalization algorithms,
including GCRMA, Mas5, dChip, and so on (see Note 4). The
differences of these methods have been discussed in previous
 Microarray Data Analysis 21

studies []. The GCRMA package takes GC content into

account when doing RMA normalization. However, one
study argued that a crucial step in GCRMA responsible for
introducing severe artifacts in the data leading to a systematic
overestimate of pairwise correlation []. Here we show the use
of RMA, but you could apply other your preferred algorithms.
To normalize gene expression using RMA algorithm, and cre-
ate an ExpressionSet object (see Note 5), type:
eset <- rma(affy.raw)

3. To save the expression data in a local file that may be used later
(to be noted, the expression values in the output are normal-
ized and log2 transformed), type:
write.exprs(eset,file="rma_norm_expr.txt")

3.1.5 Gene Annotation Gene annotation is need for further interpretation of the results.
Two Bioconductor packages are required, including Biobase [15]
and mogene20sttranscriptcluster.db [17].
1. To install and load these two packages, type:
biocLite("Biobase")
biocLite("mogene20sttranscriptcluster.db")
library(Biobase)
library(mogene20sttranscriptcluster.db)

2. The mogene20sttranscriptcluster.db package provides a variety

of detailed information for Mogene2.0ST platform, including
ACCNUM, ENSEMBL, ENTREZID, ENZYME, GENE-
NAME, GO, PATH, PFAM, PROSIT, REFSEQ, SYMBOL,
UNIGENE, and UNIPROT. To get a list of available objects in
the package, type:
keytypes(mogene20sttranscriptcluster.db)

3. To retrieve data for selected objects (e.g., ENTREZID and

SYMBOL as showed below) as a data frame, type:
gns <- select(mogene20sttranscriptcluster.db, keys(mogen-
e20sttranscriptcluster.db), c("ENTREZID", "SYMBOL"))

4. For certain types of annotations (such as gene symbol), there

could be multiple matches for the same gene. In such case, if
you only want to keep one match per gene, the most naive way
is to keep the first one. However, just skip this step if you want
to use full annotation information. To keep only the first
annotation for each gene, type:
gns <- gns[!duplicated(gns[,1]),]
Exploring the Variety of Random
Documents with Different Content
killed one down on the creek afore I came."
Mr. Tompkins took the turkey, and calling a negro boy, bade him take
it to the cook to be prepared for dinner. Then he conducted his
guest to the veranda. Uncle Dan placed his long rifle and
accoutrements in a far corner, and sat down by Mr. Tompkins.
"Wall, how's times about heah, any how, and how's politicks?" he
asked, as soon as seated.
The mountain air in America, as in Switzerland, seems to inspire
those who breathe it with love of liberty. The dwellers on the
mountains of Virginia, North Carolina and Tennessee were chiefly
Abolitionists, who hated the slave-holder as free men do tyrants, and
when the great struggle came on they remained loyal to the
Government. As a rule, they were poor, but self-respecting,
possessing a degree of intelligence far superior to that of most of
the lower class of the South.
The secret of the friendship between the planter and the hunter was
that both were, at heart, opposed to human bondage, and though
they seldom expressed their real sentiments, even when alone, each
knew the other's feelings.
Before Mr. Tompkins could reply to the mountaineer's question,
Abner and Oleah ran up to the veranda with shouts of joy and noisy
demonstrations of welcome. Uncle Dan placed one on each knee,
and for some time the boys claimed all his attention.
"Oh, Uncle Dan, you can't guess what we've got," Oleah cried.
"Why, no; I can't. What is it?" asked Uncle Dan, abandoning attempt
to return to the social chat the boys had interrupted.
"A baby! a baby!" cried Oleah, clapping his hands.
"A baby?" repeated Uncle Dan, in astonishment.
"Yes, sir; a bran new baby, just as sweet as it can be, too."
The puzzled mountaineer, with a suspicious look at Mr. Tompkins,
said: "Thought ye said the folks was all well?"
"They are," answered Mr. Tompkins, with an amused smile.
"Dinah found the baby in a clothes-basket," put in Abner.
"Oh, it's a nigger baby, is it?" asked Uncle Dan.
"No, no, no; it's a white baby—a white baby," both boys quickly
replied.
"What do the children mean?" asked Uncle Dan, bewildered, looking
from the boys to their father.
"They mean just what they say," said Mr. Tompkins. "A baby was left
at our door a short time ago in the clothes-basket by some unknown
person."
"Don't you want to see it, Uncle Dan?" Master Oleah eagerly asked.
"To be sure I do. I always liked babies; they are the perfection o'
innocence."
Before he had finished his sentence, Oleah had climbed down from
his knee, and was scampering away toward the nursery. Abner was
not more than two seconds in following him.
"Wall, now, see heah," said the hunter; "while them young
rattletraps is gone, jest tell me what all this means. Hez someone
been increasin' yer family by leavin' babies a layin' around loose, or
is it a big doll some one haz give the boys?"
"It's just as the boys say," Mr. Tompkins answered. "Some one did
actually leave a baby about six months old on this porch, and no one
knows who he was, where he came from, or where he went."
"That's mighty strange. How long ago was it?"
"About six weeks."
"Wall, now, ain't that strange? Have you any suspicion who done it?"
"Not the least."
"Wall, it is strange. Never saw no un sneakin' about the house, like?"
"No one at all."
"Humph! Well, it's dog gone strange."
At this moment the two boys, with Dinah in attendance, came out,
bearing between them little Irene.
"Here it is; here is our baby! Ain't she sweet, though?" cried Oleah,
as they bore their precious burden toward the mountaineer.
"Why it's a spankin' big un, by jingo! Ya-as, an' I be blessed ef I ain't
seen that baby before," cried Uncle Dan.
"Where?" asked Mr. Tompkins, eagerly.
Uncle Dan took the little thing on his lap, and, as it turned its large
dark-gray eyes up to his in wonder, he reflected a few minutes in
silence and then said:
"I saw a baby what looked like this, and I'll bet a good deal it is the
same one, too."
"Where did you see it?" again demanded the planter.
"That's jest what I'm tryin' to think up," said Uncle Dan. "Oh, yes; it
war in the free nigger's cabin, on the side o' the east Twin Mountain.
You know where the old cabin stands, where we used to camp when
we war out huntin'!"
"Yes."
"Wall, I war roamin' by there one day, and found two nigger men
and a woman livin' there. They had this baby with them, and I
questioned them as to where they war gwine, but one nigger, who
had a scar slaunch-ways across his face," here the narrator made an
imaginary mark diagonally across his left cheek to indicate what he
meant by "slaunch-ways," "said they war gwine to live thar. I asked
'em whar they got the baby, and they said its people war dead, and
they war to take it to some of its relations. I left 'em soon, for I
couldn't git much out o' them, but I detarmined to keep an eye on
'em. The next time I came by that way they were gone, bag and
baggage."
"The free nigger's cabin is at least twenty miles from here," said Mr.
Tompkins. "It is strange why they should bring the baby all that way
here and leave it."
"It do look strange, but I guess they war runaway niggers what had
stole the child out of spite, and when they got heah give out an' left
it. I kinder think these niggers war from the South."
"Have you ever seen or heard of them since?" asked Mr. Tompkins.
"Neither har nor hide."
At this moment a stranger to Uncle Dan came sauntering up the
lawn, and, stepping on the porch, addressed them with:
"Can you tell me where my brothers feed their flocks?"
"He's crazy," whispered Abner to the hunter. "He's crazy, and
mamma says pretend as if he was talking sense."
"Oh, they are out thar somewhar on the hills, I reckin'," Uncle Dan
answered.
Joe looked at the mountaineer for a moment, carefully examining
the hunting jacket of tanned skins, the hair of which formed an
ornamental fringe, and then said:
"I know you now. You are my Uncle Esau; but why should you be
here in Egypt? It was you who grew angry with my father because
he got your birthright for a mess of potage. You sought to slay him
and he fled. Have you come to mock his son?"
"Oh, no, youngster; yer pap and me hev made up that little fuss
long ago. I forgive him that little steal, an' now we ar' all squar'
agin."
"But why are you in Egypt? You must be very old. My father, who is
younger than you, is old—bowed down—"
"Poor boy," said Mr. Tompkins, with a sigh, "he has been a close
student, and perhaps that was what turned his head."
"Does he ever git rantankerous?" asked Uncle Dan.
"No; he is always mild and harmless."
"Have you seen my father?" Joe now asked. "He has long white hair
and snowy beard."
"No, youngster; I ain't got a sight o' the old man fur some time,"
said Uncle Dan.
"Potiphar resembles my father, but my father must be dead," and he
sank into a chair, with a sad look of despair, and, burying his face in
his hands, groaned as if in pain.
"He does that way a dozen times a day," Abner whispered to Uncle
Dan.
"It's maughty strange," said Uncle Dan, shaking his head in a
puzzled manner.
The next day, when the mountaineer was about to return to his
lonely cabin, Crazy Joe asked permission to accompany his Uncle
Esau. Consent was given, and he went and stayed several weeks.
For years afterward he stayed alternate on Mr. Tompkins' plantation
and at the home of the mountaineer.
 CHAPTER V.
THE MUD MAN.

Sixteen years, with all their joys and sorrows, all their pleasures and
pains, have been numbered with the dead past. Boys have grown to
be men, men in the full vigor of their prime have grown old, and
creep about with bent forms and heads whitening, while men who
were old before now slumber with the dead. Girls are women, and
women have grown gray, yet father Time has touched gently some
of his children.
Abner and Oleah Tompkins are no longer boys. Only the memory is
left them of their childhood joys, when they played in the dark, cool
woods, or by the brook in the wide, smooth lawn. Happy childhood
days, when neither care nor anxiety weighed on their young hearts,
or shadowed their bright faces.
Abner is twenty-five—a tall, powerful man, with dark-blue, fearless
eyes, light-haired, broad-chested and muscular.
Oleah, two years younger, and not quite so tall, is yet in physical
strength his brother's equal. He has the dark hair and large, dark,
lustrous eyes of his Southern mother.
The brothers were alike and yet dissimilar. They had shared equally
the same advantages; they had played together and studied
together. Playmates in their childhood, friends as well as brothers in
their young manhood, no one could question a doubt of their
brotherly love. Where one had been, the other had always been at
his side. No slightest difference had ever yet ruffled the smooth
surface of their existence. Yet they were dissimilar in temperament.
Abner was slow and cool, but perhaps more determined than his
brother, and his reason predominated over his prejudice. Oleah was
rash, impetuous and bold, and more liable to be moved by prejudice
or passion than by reason. Abner was the exact counterpart of his
Northern father, Oleah of his Southern mother.
Their political sympathies were different as their dispositions.
Although of the same family, they had actually been taught opposite
political creeds—one parent in a half-playful way, unconsciously
advocating one idea; the other as firmly and unconsciously
upholding another, and it was quite natural that the children should
follow them. But this difference of opinion had bred no discord.
Sixteen years have wrought a wonderful change in Irene, the
foundling. Her parentage is still a mystery, and she bears the name
of her foster parents. She is just budding into womanhood, and a
beautiful woman she promises to make—slender and graceful, her
small, shapely head crowned with dark brown hair, her cheeks
dimpling with smiles, mouth and chin firm and clear-cut and large,
dark-gray eyes beneath arching brows and long silken lashes filled
with a world of tenderness.
Irene could not have been loved more tenderly by the planter and
his wife had she been their own child. They lavished care and
affection upon her and filled her life with everything that could
minister to her comfort and delight, and every one knew that they
would make generous provision for the little waif who had gained so
sure a place in their hearts.
Sixteen years had made some change in the planter. His hair had
grown whiter, his brow more furrowed with care, and he went about
with a heavy cane; yet he was vigorous and energetic. He had
grown more corpulent, and his movements were less brisk than of
yore. Father Time had dealt leniently with his wife. Her soft, dark
hair was scarcely touched with silver; her cheeks were smooth and
her eyes were still bright and lustrous. Her voice had lost none of its
silver ring, her manner none of its queenly grace.
No ray of light had pierced the darkened mind of Crazy Joe. All these
long, weary years he had been waiting, waiting, waiting, for his
father Jacob to come down into Egypt, but he came not. He still
talked as if it was but yesterday that he had been cast into the pit by
his brethren, and then taken out and sold into Egypt. He spent his
time in turns at the planter's and Uncle Dan's cabin. He was well
known throughout the neighborhood, and pitied and kindly treated
by all. His strange hallucination, although causing pain and
perplexity to his shattered mind, worked no change in his gentle
disposition; his sad eyes never flashed with anger; no emotion
varied the melancholy monotone of his voice. When at the home of
the planter, Joe divided his time between the stables, the garden
and the library. He would have been a constant reader of the Bible,
Josephus, Socrates, Milton's "Paradise Lost," had it not been
discovered by Mrs. Tompkins that these books only tended to
increase the darkness in which his mind was shrouded, and she had
them kept from him. At Uncle Dan's mountain home he passed his
time in hunting and trapping, becoming expert in both.
Sixteen years had wrought a great change in Uncle Dan, bowing his
tall and sinewy form. His face, which he had always kept smooth
shaven, had grown sharper and thinner, and his long hair hanging
about his shoulders, had turned from black to gray; yet his eye was
as true and his hand as steady as when, in his youthful days, he
carried away the prize at the shooting match. His visits to the
plantation became more frequent and his stays longer, for the old
man grew lonesome in his hut, and he was ever a welcome guest at
the Tompkins mansion.
Sixteen years had made a wonderful transformation in the politics of
the country. The Whig party had been swallowed up by the
Republican or Abolition organization. The seeds of freedom, sown by
Clarkson, Brown and others, had taken root, and, in the Fall of 1860,
bade fare to ripen into a bounteous harvest. The Southern feeling
against the North had grown more and more bitter, and the low,
rumbling thunders of a mighty storm might have been heard—a
storm not far distant, and whose fury naught but the blood of
countless thousands could assuage.
"In the beginning, God created Heaven and the earth, and all that
was in them, in six days, and rested on the seventh."
The speaker was Crazy Joe, the time, midsummer of 1860, the place
the banks of a creek at the foot of the mountains, not more than
two or three hundred feet from Uncle Dan's cabin.
"Then the book says God made man out of clay. Josephus says he
called the first man Adam, because Adam means red, and He made
him out of red clay. Now, if man could once be made out of clay,
why not now? Maybe God will let me make a man, too."
Filling his hands with mud, he set vigorously to work. No sculptor
could have been more in earnest than was Crazy Joe. He rolled and
patted the mud into shape, first the feet, then the legs, then the
body. Occasionally the body would tumble down, but he patiently set
to work again, persevering until he had body, arm and head all
completed. His mud man was a little over five feet in height, and
greatly admired by his maker and owner.
"Now I have accomplished almost as much as God did," soliloquized
Joe. "I have made a man of clay; it only remains for him to speak
and move, and he will be equal to any of us."
He went to the cabin and acquainted Uncle Dan with the wonderful
work he had performed, and asked him to come and see it. The next
day he went to view the object of poor Joe's two days' labor, greatly
to Joe's delight. Uncle Dan then returned to his cabin for his gun,
and Joe went to Snagtown, which was between Mr. Tompkins'
plantation and the hunter's cabin.
Joe there informed the storekeeper, the village postmaster, and a
few others, of his remarkable piece of handiwork, and asked them to
come and see it. They promised to go the next day, if Joe would stay
all night in the village.
Joe stayed, and that night there came a heavy rain. The creek
overflowed and Joe's mud man was washed away. He conducted a
party of hunters to the spot next morning, but the man of clay had
vanished.
"He must have walked away," said Joe shaking his head in a puzzled
manner. "He has gone off, though I cautioned him to wait until I
came back."
The hunting party explained to Joe that his mud man had become
tired of waiting, and left, and went off themselves, leaving the
mortified Joe searching about the soft soil for tracks of the missing
mud man. His search for the trail took him to Snagtown.
Patrick Henry Diggs, whom we met in his boyhood as the youthful
orator at Mr. Tompkins' was, in 1860, a lawyer. His parents were
dead, leaving him a limited education, a superficial knowledge of
law, and a very small property. The paternal homestead was
mortgaged, but Mr. Diggs still kept old Mose, for the sake of being a
slaveholder and maintaining aristocratic appearance. Mr. Diggs had
but little practice, and found it a difficult thing to make his own
living. He was about twenty-eight years old, short and plump like his
father. The most peculiar portion of his anatomy was his head. The
forehead was low, and the small round head more nearly resembled
a cocoanut painted white, with hair on its top, than anything else to
which we can compare it. The hair was very thick and cut very short.
The eyebrows were heavy and close together, the eyes dark gray
and restless, the nose small and straight. The most admirable
portion of his physiognomy, Mr. Diggs thought, were his side-
whiskers, which were short and dark, growing half-way down his
small, red cheeks and coalescing with his short mustache. Mr. Diggs
was exceedingly aristocratic, and wore gold-rimmed spectacles on
his short nose. These glasses, which gave him a ridiculous
appearance, were removed when he wanted to read or exercise his
unobstructed vision. His friends tried to persuade him to give them
up, but in vain. And with his glasses on his nose, his head thrown
back in order to see persons of ordinary height, and his fat little
hands in his pockets, he strutted about the streets of Snagtown.
Mr. Diggs, like his father, was a politician. In the campaign of 1860
he was a candidate for the district attorneyship of his county. His
dingy little office, with its scant furniture and exceedingly small
library, was deserted, and he spent most of his time on the streets,
discussing the political issues. On the day that Crazy Joe was in
search of his mud man, Mr. Diggs, as usual was strutting about the
streets, his hands in his pockets, his glasses mounted on his nose,
wherefrom a very evident string extended to his neck.
"I tell you," said Mr. Diggs, closing his little fat right hand and
striking therewith the palm of his little fat left hand, "I tell you, sir, I
—I do not favor outlawry, but I do believe one would be doing our
country a service by hanging every man who votes or attempts to
vote the Abolition ticket."
"Oh, no, Mr. Diggs," said Abner Tompkins, who chanced that day to
be in Snagtown, and overheard the remark; "the ballot is a
constitutional privilege, and no man should be deprived of his right."
"Yes—ahem—ahem! but you see, when there is a man on the track
who, if elected, will set all our niggers free, we should object. You
know—no, you don't know, but we lawyers all know—that private
property can not be taken for public use without a just
compensation, and still the Abolition candidate will violate this
portion of our constitutional law."
"You don't know yet; Mr. Lincoln has not yet declared what he will
do," replied Abner.
"Has not? Hem, hem, hem!" Mr. Diggs stumped about furiously, his
head inclined backward in order to see his companion's face through
his ornamental glasses, while he cleared his throat for a fresh burst
of thunder. "Has not, hey? Hem, hem! He might as well. We all know
what he will do if elected. And I'll tell you something more," he
added, walking back and forth, his hands plunged in his pockets,
while seeming to grow more and more furious, "if Lincoln is elected
there will be war!" (Great emphasis on the last word.)
At this moment Crazy Joe, who had reached the village in search of
his mud man, came up to the excited Diggs, and, laying his hand on
his arm, in a very serious voice said:
"Say, why didn't you stay where I put you until I showed you?"
"What do you mean?" demanded Mr. Diggs, pausing in his agitated
walk, and gazing furiously into the lunatic's face, for he suspected
some one of attempting to play a joke on him.
"What made you go away before I showed you?" said Joe, earnestly,
gazing down upon the furious little fellow.
"I—I don't understand what you mean," said the puzzled Mr. Diggs,
drawing himself up to his full height, which was hardly imposing.
"When I make a man of mud, and go off and leave him, to get
people to come and look at him, I don't want him to go off, as you
did, before I come back."
Abner Tompkins, and several others, who had heard the story of
Joe's mud man, were now almost bursting with suppressed
merriment.
"I can't tell what the deuce you mean?" said the angry Mr. Diggs.
"I made you out of mud and clay, and left you standing by the big
tree at the creek while I went to get some people to show you to,
that I might convince them that man was made out of clay, but
before I got back you walked off. Now, why didn't you stay until I
showed you?"
The men gathered about Mr. Diggs could no longer restrain
themselves, and burst into peals of laughter, which made Mr. Diggs
furious.
"This is some trick you are playing," he cried, and, turning upon his
heel, he strutted away to his office, where he shut himself up for the
next two hours.
The joke spread rapidly, and in two hours every one in the village
knew that Crazy Joe claimed Mr. Diggs as his mud man; while poor
Joe, satisfied that he had found the object of his creation, consented
to go home with Abner.
 CHAPTER VI.
A TRANSITION PERIOD.

All Snagtown was astonished one day when a flaring handbill

announcing that Abraham Lincoln and Stephen A. Douglas would
speak in that unpretentious little village. Their presence there was
due to the accident of missing connections in passing from one city
to another.
It would have been hard to say whether the citizens of Snagtown
were more astonished or indignant. A public meeting was called the
day before the Abolitionists were advertised to speak, to determine
what means could be taken in this emergency. The Mayor presided,
and the residents, not only of the village, but of all the surrounding
country, were urged to be present.
"I tell you, gentlemen—hem! hem!—it will never do," said Mr. Diggs,
as he strutted about, his glasses on his nose, casting upward
glances into the faces of those who were discussing the question.
"Hem! hem! hem! I tell you it will not do at all," and he expectorated
spitefully upon the pavement. "We must prevent Lincoln's speaking
here, if we have to mob him. He comes not only to deprive us of our
slaves, but to destroy the flag of Washington and Marion, the
glorious Stars and Stripes! I, for one, am in favor of saying he shall
not speak."
"So am I," said another.
"And so am I," said a third.
"And I, and I, and I," came responses from many voices.
"Hem, hem, hem!" began Mr. Diggs, shrugging his shoulders, and
moving about furiously, indicating thereby how much in earnest he
had become. "I tell you we must not permit it. Why, it's treason. Yes,
sir; he teaches treason, and it's our duty, as law-abiding citizens, not
to permit him to speak."
"Well, now, do you make them pints, when we have our meetin' to-
morrow night," said an illiterate Virginian.
"Hem, hem, hem!" began Mr. Diggs, thrusting his hands deep into
his pockets, his head on one side, kicking his feet alternately one
against the other. "I will. Hem, hem! I am going to make a speech
just about an hour long—ha! ha! ha!—so that no one else will get a
chance to put in a word, and we shall have it all our own way." The
young lawyer, highly pleased with the favor that he flattered himself
he was gaining politically, finished his sentence with a gleeful
chuckle, and strutted about, swelling with his own importance.
All over the village could be seen groups of men, from five to twenty
in number, discussing the propriety of allowing "Abe Lincoln" to
speak in the village. A majority seemed opposed to it, and a few of
the more reckless spirits talked of tar and feathers and fence rails.
The evening for the public meeting, which was to decide the all-
important question, arrived. The town hall was crowded to its
utmost capacity. Mr. Tompkins and his two sons were present, and
so was Uncle Dan, the mountaineer. The meeting was called to order
and the Mayor took the chair. He was a man past the meridian of
life, a slaveholder and a royal Southerner. The long, white beard
falling down upon his breast gave him a patriarchal look.
The uproar and confusion of tongues were hushed, and all awaited
the speaker in anxious silence.
A call was made on any one present to state the object of the
meeting. A man sprang at once to his feet, and succinctly informed
the chairman that the "object of this meetin' is to determine the
question whether or not it is best to 'low Abraham Lincoln, the great
Abolitionist, to speak in the town. I believe them's all the pints to be
discussed," and he sat down. Another and more voluble speaker
arose and addressed the meeting. He was of the class called "fire-
eaters," and was strongly and directly opposed to Lincoln's visit to
Snagtown. His speech was replete with the vilest vituperations his
brain could conceive, or his tongue utter, against the Republican
party. He regarded them as robbers, as enemies who should be shot
down at sight, and he was in favor of greeting Abe Lincoln with tar
and feathers if he dared show himself in Snagtown.
Several others spoke in the same vein, and then Mr. Diggs rose. His
speech of an hour proved not half so long. It was full of empty-
sounding words and borrowed ideas, for there was little originality
about Mr. Diggs.
All, so far, had been against the proposed debate between Lincoln
and Douglas, but now a man rose in the audience whose word
always carried weight. It was Mr. Tompkins, the planter.
"Mr. Chairman," he began, in even, modulated tones, "I am, indeed,
surprised that men of intelligence should give vent to such
expressions and such feelings as we have heard this evening—men
who know the law, and claim to be law abiding citizens. Are we
savages or border ruffians, that we must be swayed and controlled
by mob law? Have we not a Constitution and Constitutional
privileges? Have we not statute laws to protect us against wrongs
which others may inflict? Then why resort to mob law? Why disgrace
our fair State and put the blush of shame on all good citizens by
attacking, like outlaws, a stranger among us? Our Constitution gives
to all freedom of speech, and we have no right to deny any man this
Constitutional privilege."
Mr. Tompkins proceeded quietly but forcibly, pointing out to the
malcontents the error of their plans. In conclusion, he said:
"I may be the only one in the house who opposes these views, but
as one I say this, though I be alone. I will oppose with violence the
attempt to injure Mr. Lincoln. You are not compelled to vote for him,
even to hear him speak; but if Mr. Lincoln comes here, by Heaven!
he shall speak."
"So say I, an' I swar if any sorry hound attempts the mobbin'
business, he'll have to cross my carcass fust." The speaker was
Uncle Dan, and as he spoke he drew up his tall figure by the side of
Mr. Tompkins, holding his ominous-looking rifle in his hand.
Abner also rose and took his place at his father's side, but Oleah
kept his seat. This was the first visible difference of opinion between
the brothers.
Several who had been emboldened by Mr. Tompkins' words now
declared that they thought it best not to oppose Mr. Lincoln's
speaking there, as it would increase his popularity in other localities.
One or two of the more fiery replied, maintaining that their case was
beyond the remedy of civil law; that mob law was the only law which
should be meted out to scoundrels and Abolition thieves, and if
some of the citizens intended to espouse the cause of Abe Lincoln,
and fight for him, now was as good as any to settle the matter. A
riot seemed inevitable, but a laughable event now happened,
changing anger into mirth.
Mr. Diggs, fearing that his legal knowledge would be called into
question, now rose and said:
"I wish to make one other statement, in order to put myself right
before the people. I knew the Constitutional law referred to by Mr.
Tompkins, giving every man freedom of speech, and I can give you
the book and the page—"
"Oh, you need not," said a wag in the audience. "Answer this
question instead: Are you Crazy Joe's mud man, and why did you
leave before he came back to exhibit you?"
"Oh, stop that nonsense! I came here to talk sense, not to hear of a
fool's ravings," cried the indignant Mr. Diggs.
But everybody had heard the story of the mud man, and hostile
feelings now gave way to laughter. The laugh was kept up until Mr.
Diggs became enraged and left the assembly, swearing that they
were "all a pack of fools."
A compromise was effected. Mr. Lincoln and Mr. Douglas were to be
permitted to speak in a grove near the village, but not in the village
itself. The next day Mr. Tompkins and Abner, and a few others, with
the aid of their negroes, erected a speaker's stand, and arranged
seats for an audience of over two thousand persons. There were still
low murmurs of discontent, but the most bitter malcontents had
been overawed by the firm stand taken by Mr. Tompkins. Many
others had caught his spirit, and defied the hostile threats of the
opponents of free speech.
The occasion had been so thoroughly advertised by the meeting and
the threats and opposition of those who wanted to prevent it, that
the whole country for miles around turned out. People on foot, on
horseback, in carriages and in wagons, came until thousands were
assembled on the spot, many prompted by curiosity to see the bold
Abolitionist who dared invade the sacred soil of Virginia and
propound his infamous doctrine.
About ten o'clock two carriages rolled in from the nearest railroad
station, bearing the two disputants, with friends of each in
attendance. There was an eager craning of necks, and a hushed
whisper went through that vast audience as the two opponents for
the highest political honors of the country descended from the
carriage.
"Who are they?" "Where are they?" "Is that big, two-hundred-and-
fifty-pounder Douglas?" "Is that short, stout-built man with big
burnsides Lincoln?" and a hundred other questions of a like
character were asked.
A few preliminaries were arranged. Mr. George Washington Tompkins
was chosen chairman, and took his place on the stand. Two New
York reporters were present with note-books and pencils.
The first speaker introduced was Mr. Stephen A. Douglas. His speech
—eloquent, patriotic and straightforward—generously concluded with
an exhortation to the audience to listen calmly, without any
expression of bitterness, to his opponent, who chanced to differ
from him on the great question of the day. When Mr. Douglas took
his seat, Mr. Tompkins rose and introduced Mr. Abraham Lincoln, a
tall man, wearing short, dark whiskers on his chin, and with hair
slightly streaked with gray.
A subdued hiss from many lips was heard as the great "Abolition
candidate" arose.
After a smile as of compassion upon his audience, Mr. Lincoln began
speaking. He talked mildly and candidly, yet freely, notwithstanding
the feeling evinced by some of his hearers. Those deep, rich tones
rang through the surrounding grove as he clearly and forcibly
expounded the principles of the Republican party, showing them to
have been either misunderstood or misrepresented by his opponent.
Many who had come to prevent the hated Abolitionist from speaking
now listened with interest. This was not such iniquitous doctrine
after all. Every point made by Mr. Douglas was successfully met, and
his own argument arrayed against him. Mr. Lincoln spoke for two
hours, and at the conclusion of his address his bitter enemies were
forced to admit that he was a man of immense power. His oratory
was so grandly sublime in effect that when he took his seat an
outbreak of applause, which could not be suppressed, could not be
restrained, burst from the spell-bound audience.
Mr. Tompkins went to the meeting a Douglas man, but he left with
the full determination to vote for Abraham Lincoln at the coming Fall
election, as did Uncle Dan and many others. This was truly a
transition period, as the whole world was to learn in a few short
months. The Whig party was dwindling away, and slavery was
withered and scorched before the fiery eloquence of Lincoln,
Sumner, and other similar orators. Freedom was dawning, but it was
to be ushered in with fire, and sword, and death.
Mr. Tompkins and his sons were late in coming home that evening.
Abner and Oleah sat side by side in the family carriage, yet neither
spoke. Hitherto, every event had been fully discussed; every feeling
shared by the brothers; but a silence that was almost coolness now
sealed their lips. A thousand conflicting thoughts swept through their
minds.
Abner was convicted, converted, by the new doctrine to which he
had listened, and the melodious voice of the orator was still ringing
in his ears as the carriage rolled homeward. He still seemed to see
the tall, rugged form and plain face, lit up with something rarer than
beauty by his eloquent pleading for four millions of enslaved human
beings.
Oleah was in a gloomy mood. He had listened with angry impatience
to the exposition of views so different from his own, and that his
father should have presided over the meeting, and stood openly side
by side with the Abolitionist, stung his Southern prejudices and
vexed him to the soul.
The trio were driven home in silence, and parted for the night,
without any reference to the events of the day.
At the table the next morning the discussion of the day before was
first alluded to. Mr. and Mrs. Tompkins, Abner and Oleah, sat for
some moments in silence—a silence both painful and awkward, and,
in this family circle, unusual; but Irene entered the breakfast room,
bright and unconscious, eager to know all that had passed at
Snagtown the day before.
"We heard an excellent speech," said Abner.
"Yes; Douglas did well," put in Oleah.
"I mean Mr. Lincoln," said Abner. "Douglas' speech was good, but his
position was entirely demolished by Mr. Lincoln's eloquent
reasoning."
"You don't call the harangue of that contemptible old demagogue
reasoning, do you?" asked Oleah, astonished and indignant.
"I certainly do," replied Abner. "His reasoning appeared to me clear,
and his conclusions logical."
"And I," cried Oleah, laying down his knife and fork in his
excitement, "I declare I never before heard so much sophistry, and
not very plausible sophistry, either."
"You are prejudiced," said Abner, coolly.
"It is you who are prejudiced. Why he actually asserted we would be
more prosperous if there was not a slave in the United States."
"Yes, and proved his assertion," said Abner.
"Oh, you let him pull the wool over your eyes." There was a sneer in
his voice. "I tell you there was neither logic nor reason in what he
said. No logical conclusions can be drawn from false premises; no
assertions can stand unsupported by proof."
"What did he assert that he did not prove?" asked Abner.
"What did he prove that he asserted?"
"You evade my question by asking another."
"Precisely the same plan Mr. Lincoln adopted," replied Oleah.
"You are prejudiced against Mr. Lincoln, Oleah. Now, tell me what he
said that any fair-minded man in the world can not agree to?"
"He said that slavery should not wither and blight another inch of
territory if he could help it."
"What objection can even a believer in slavery have to that? We
have an immense scope of country where slavery is permitted; then
why extend it to Territories where it is unpopular?"
"But can you not see what lies in the background?" said Oleah,
bitterly. "Mr. Lincoln lifted the curtain high enough for one who was
not blinded by his eloquence to see what was behind it. I would not
fear to wager everything I own that Mr. Lincoln, if elected, will set
free every slave in the United States, before he has been in the
presidential chair a twelvemonth."
"Did he not say that such emancipation would be unwise policy?"
"He said so, but his tone and manner belied his words."
"Confess now, Oleah, that you are a little prejudiced against Mr.
Lincoln," said the father, good-humoredly.
"You may call it prejudice or what you like, father," Oleah answered,
his flushed face showing how deep was his feeling; "but if Mr.
Lincoln is elected you will not have a nigger when his term is over, if
he should be permitted to take his seat."
"Why, my son, you can't think he would not be permitted to take his
seat?"
"That is a question, father. Each State has its rights. Southern people
have rights, and rather than be cheated of them they may resort to
force."
"Now, Oleah," said Abner, "you don't for a moment suppose that if
Mr. Lincoln should be chosen President by the voters of the United
States, that any considerable body of intelligent people could be
found who would be unfair enough, or foolhardy enough, to attempt
to prevent him from taking his seat?"
"I certainly do," answered Oleah, with an air of conviction.
"You are a Democrat; do you not hold with us Democrats that the
majority should rule?"
"That has nothing to do with it," said Oleah, hotly. "The North and
the East outnumber the South, and they have formed a combination
for her ruin, and the impoverishment of her people. They have
nothing at stake in Lincoln's election; we have everything. They have
nothing to lose—we, all. Our interests conflict. They see an opulent
and growing South, and have set their inventive Yankee genius at
work to compass its ruin. Our cotton fields, our rice fields, our sugar
crops, our tobacco crops, are the production of slave labor, and the
abundant wealth of the South excites the emulation of the cold and
envious North. If they can deprive us of this slave labor, they will
have killed the goose that lays our golden eggs, and may surpass us
in wealth and power. This they have determined to do. They have
tried it by legislation, and so far have failed. They outnumber us in
votes, because there every worthless fellow's vote counts as much
as that of a Governor or a man who owns a thousand slaves. How
can they accomplish our ruin? By electing as president a man whose
every breath is poison to slavery; a man who may, at any time,
under the fancied exigencies of the moment, declare all slaves free.
Their plans are deep and shrewd, but there are heads in the South
as wise as theirs, and eyes that can see the danger in time to avert
it."
"You are crazy, Oleah," said Abner; "your very words are treason."
"If treason, then his mother is infected with the same disease, and,
in the language of Patrick Henry, 'If this be treason, make the most
of it,'" said Mrs. Tompkins, with a laugh, in which all joined.
"I am sure we ought to get at the truth of this question," said Mr.
Tompkins; "we have both sides represented."
"Who will judge between us?" asked Mrs. Tompkins.
"All have taken sides except Irene. Which side are you on?" asked
Oleah.
"I know nothing about either side," the girl answered, lightly; "so
how can I choose?"
Mrs. Tompkins' love for her sunny land was next in her heart to her
love for her husband, and forced her to espouse a cause which, to
her, seemed patriotic. This was the only question on which she and
her husband differed, and it was avoided by both as much as
possible, yet sometimes, in spite of their precautions, it would creep
into their family conversations.
"Irene is the proper one to act as judge," said Abner.
"Why?" Irene lifted her eyes in wonder.
"Because you know nothing about it."
"Do they make the best judges who know the least?"
"Frequently; and a juror who knows anything of the case he is to
pass a verdict on is incompetent, so you are a competent juror, any
way, Irene; and as one woman is equal to twelve men you can
complete the entire panel."
"I beg pardon of the court," said Irene, rising from the table, "but I
can not sit on this jury. I am prejudiced on both sides. I have friends
on both sides, and I could not render an unbiased verdict."
"That's no excuse," said Abner.
"If it's not, the new piece of music you bought me is, so I leave you
to your discussion, and hope you may effect a happy compromise."
She was gone.
There was a moment's silence, and then the rippling music of her
voice filled the halls and rooms of the great house.
"I wish the name she bears was rightfully hers, though I am glad
she is not my sister," Abner said to himself. The same thought
flashed through Oleah's mind, and, as usual, the mobile face
betrayed his thoughts. Every one seemed always to understand his
feelings.
Irene had just returned from school, an accomplished beauty and an
acknowledged belle.
No wonder strange emotions stirred the hearts of the brothers, and
that thoughts gained entrance in their breasts which might prove
more disastrous than mere political differences.
 CHAPTER VII.
THE ELECTION AND THE RESULT.

The election of 1860 was an exciting one. No means were spared to

poll every possible vote. Lincoln was the Republican candidate,
Douglas a Northern, and Breckinridge a Southern Democrat, and Bell
the Whig and "Know-Nothing" candidate, and all four parties worked
vigorously.
Mr. Tompkins and his sons reached Snagtown early in the morning.
The village was already alive with the stir and excitement. The polls
opened at sunrise, and men were soon crowding around them,
quarreling, disputing, joking. The morning air was crisp and frosty,
and the people were compelled to walk about briskly to keep from
being chilled.
A dirty faced urchin, with a pumpkin under one arm and some
turnips under the other, paused in front of the polls, and, stretching
out his neck like a young rooster achieving his first crow, bawled
out:
"Hurrah for Douglas!"
It was the first patriotic wave which had caused an undulation of his
infantile breast.
There chanced to be another boy, more dirty than the first, sitting on
a fence near by gnawing an apple-core. His "pa" was a Breckinridge
man, and, regarding this outburst as a challenge, he threw away the
apple-core and fell with fury upon him of the pumpkin and turnips.
Coming head first into the stomach of the Douglasite, he sent boy,
pumpkin, and turnips into the gutter.
The enraged young Douglasite scrambled to his feet, and, leaving
his vegetables behind, started in hot pursuit of the now fleeing
Breckinridgeite, while shouts and cheers went up from the many
spectators.
Mr. Diggs came along, engaged in conversation with a farmer whom
he was trying to persuade to vote for himself and Breckinridge, for
Mr. Diggs was a candidate for the office of District Attorney. On
account of his small stature, the candidate was compelled to walk
with upturned face, in order to watch the effect of his words upon
the tall Virginian. The sidewalk being crowded, they had taken the
middle of the street, and Mr. Diggs struck his toe with such force
against the abandoned pumpkin that he was thrown down, and,
falling on the pumpkin, he rolled with it into the gutter, which was
half full of mud and water. Shouts and yells of laughter greeted Mr.
Diggs as he scrambled to his feet and picked up the glasses which
he had lost in his fall.
"By jingo, Diggs, ye look like Crazy Joe's mud man now!" cried some
one from the crowd.
This was too much for the candidate, and, with something very
much like an oath, he hurried away to change his clothes.
As the day advanced, the crowd increased, and as electioneering
progressed, the crowd became very noisy.
There was Mr. Snag, a direct descendant of the founder of
Snagtown, who claimed political honors. He was a candidate for
County Judge. He had been one of the pioneers, had fought Indians,
bears, wolves, panthers, and rattlesnakes, to establish this growing
country. He had always been the workingman's friend, and was now
ready to sacrifice himself on the official altar.
Mr. Snag had been a clothing merchant, noted for close dealings
with his customers and oppression of his employes; but two or three
months before he announced himself a candidate, a change came
over him. His harshness of voice and manner grew subdued. He
became not agreeable only, but accommodating and charitable. He
attended church and the bar-rooms regularly, and was developing
into a general favorite. He was welcomed in the most select circles,
yet he was not exclusive. No man was too ragged, too dirty, or too
drunk to cause Mr. Snag to be ashamed of his society. He was more
than changed; he was completely metamorphosed.
On election day he was more affable than ever. He was at hand to
lift up a drunken rowdy who had fallen over the pumpkin, and led
him at once to the voting place, to poll his vote for himself and
Breckinridge. But the pumpkin remained.
Later in the day, two rowdies, from the country, having imbibed too
much of the electioneering beverage, got in a quarrel. One struck
the other, and he fell by the pumpkin. A friend of the fallen man
seized the pumpkin, and broke it into fragments over the other
man's head, bringing him to the ground, of course. A general melee
was averted only by the appearance of some good-natured
candidate, who tried to restore peace, followed by a couple of
constables, who at once arrested the malcontents.
In the afternoon Abner and Oleah went up to the polls. The two
brothers had been silent during the forenoon, both seeming to avoid
the political question which was agitating the Nation.
"Who are you going to vote for, Abner?" asked Mr. Diggs, strutting
up to the young planter with a smile he thought becoming a District
Attorney. "Is it Breckinridge, Douglas, or constitutional unionist Bell?"
"Neither," Abner answered.
"Who, then, is your man?" asked the inquisitive Mr. Diggs, thrusting
his hands deep into his pockets, and tipping first on his heels then
on his toes, as he looked up, with an engaging smile, into the face of
the man before him.
"I shall vote for Abraham Lincoln," Abner answered firmly.
"Pshaw! you are joking," said Mr. Diggs, his little eyes twinkling
idiotically behind his glasses.
"I was never more in earnest."
"Why, man, they'd hang you if you voted for Lincoln!"
"I shall risk it, at all events."
His brother's words brought a sharp pain to Oleah's heart. He
stopped suddenly, and laid a detaining hand on Abner's arm.
"Abner, you surely do not intend to vote for that Abolitionist?" he
said, with a ring of defiance in his voice.
"I do," was the firm reply.
"For heaven's sake, think what you are about. Do you want to ruin
the country?" Entreaty and distress was melting his indignation.
"No, I want to save it," was the calm reply.
"How can it be that you will vote for an abolitionist?"
"Because his principles and mine are the same," said Abner,
earnestly.
The brothers were nearer a quarrel than they had ever been in their
lives. Oleah's feelings were wounded, and he turned away, leaving
his brother to go his way alone.
But three votes were polled in Snagtown for Abraham Lincoln, and
Abner Tompkins, his father, and Uncle Dan, were supposed to have
cast them.
Late that evening Mr. Tompkins and his sons rode home. The trio
were silent and thoughtful, but they little dreamed what that day's
work would bring forth.
Great was the consternation of the Southern leaders when the result
of the election became known. Reports were fluctuating from the
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