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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

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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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
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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.
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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
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RNA-Seq data without a reference genome. 10. Bevilacqua CB, Basu S, Pereira A et al (2015)
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
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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
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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
13:64 20. Leng N, Dawson JA, Thomson JA et al (2013)
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babraham.ac.uk/projects/fastqc/ 21. Anders S, Huber W (2010) Differential expres-
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 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.
Another Random Scribd Document
with Unrelated Content
was running off my face and I could feel it on my back, too. With a
little wind blowing across from the woods and Sugar Creek I felt fine
even in the hot sun. I certainly wasn’t getting tired as fast as I
thought I would on account of when a boy sweats at hard work and
the wind blows a little, he feels better than when he just kinda lazies
around and tries to keep cool.

I wished Pop, who had gone somewhere for something, would

hurry home and see me working hard. It was almost fun hoeing the
potatoes, though it was kinda hard not to stop at the end of each
row and pick and eat a few luscious blackberries which grew there.
In fact, I did stop a few times, which is maybe why I got to the end
of each row quicker.

Once I got thirsty, and went into the house for a drink of water,
and Mom called out to the kitchen from the front room and said,
“That you, Theodore?” which is Pop’s first name.

“Nope, it’s just me,” I said to Mom.

“Come on in a minute, Bill. Somebody wants to see you.”

“Who?” I said, wondering who it was and hoping it wasn’t

anybody I didn’t know.

I peeked around the corner of the kitchen door and saw our lady
Sunday school teacher. All of a sudden I felt good, although kinda
bashful, on account of I was in my overalls and was probably very
dusty and sweaty and maybe had my hair mussed up.

We said a few bashful words to each other, and she said, “I

brought you that book of Indian stories,” and right away I was
thinking of little “Snow-in-the-face” up North and wishing I could go
up and see him again.

I thanked her for the book, and said, “Well—thanks, that’s swell—I
mean, Thank you so very much,” which was what I thought Mom
would want me to say in the way I said it.

“Don’t overwork,” she said to me with a smile in her voice, and I

said “I will,” and was going out the kitchen door before I knew I’d
said the wrong thing. She certainly was a good Sunday school
teacher, and knew how to make a boy like her, and also want to
come back to Sunday school every Sunday.

Just as I was about to let the door shut behind me quietly like I do
when we have company, I heard the news on the radio in the front
room, and I knew that maybe Mom and my teacher had been
listening to the radio when I came in, and had turned it low for a
jiffy. One of the things I heard was about a little St. Paul, Minnesota
girl named Marie Ostberg having been kidnapped and a reward
being offered by the father... Then I heard the announcer mention
something that I thought was a wonderful idea and it was: “Duluth—
the hayfever colony—will have thousands of new visitors this year,
because the heavy rains throughout the nation have made it the
worst for pollen in many years. Thousands will be going north...”

That would give us two reasons why some of the gang ought to
get to go—overwork and hayfever. Dragonfly had the hayfever, and if
I worked awful hard, I might overwork, although it’d be easier to
have hayfever if I could only get it.

Right that second, while I was picking up the hoe to go back to

the potatoes again, I heard our car horn and Pop was at the gate,
waiting for me to come and open it. Boy, was I ever glad I was hot
and sweaty and that there were four or five long rows of potatoes
already hoed which Pop could see himself.

“Hi,” I said to my reddish-brownish-mustached Pop. And he just

lifted one of his big farmer hands and saluted me like I was an
officer in the army and he only a private. I swung open the gate,
and, seeing the gladiolus by the mail box, stopped and took three or
four quick deep sniffs at them, just as Pop swung inside and stopped
beside the big plum tree in the gravelled driveway.

Then I looked quick at the sun, to see if I could sneeze, and I

actually did, three times in quick succession, just as Pop turned off
the motor and heard me do it.

“I hope you aren’t going to catch cold,” Pop said, and looked at
me suspiciously. “You boys go in swimming today?”

“The water was almost too hot,” I said. “I never felt better in my
life, only——” Right that second, something in my nose tickled again
and I sneezed and was glad of it. “Maybe I’m allergic to something
down here....”

“Down where?” Pop said, and looked at me from under his heavy
eyebrows, which I noticed weren’t up any more but were starting to
drop a little in the middle, like he was wondering “What on earth?”
and trying to figure me out, like I was a problem in arithmetic or
something.

“I mean——” I started to answer him, and then decided maybe it

was the wrong time to talk to Pop about what I wanted to talk to
him about. So I said, “Well, I better get back to those potatoes.
There are only two more rows.”

“Back to them?” Pop said, astonished. “You mean——?” He slid

out of our long green car and looked toward the garden, and even
from where we were, you could see that somebody had been hoeing
the potatoes. “Well, what do you know about that? That’s wonderful!
That’s unusual! That’s astonishing,” which I knew was some of Pop’s
friendly sarcasm which he was always using on me, and I sort of
liked it on account of Pop and I were good friends as well as he
being my pop and I his red-haired, freckled-faced overworked boy,
who didn’t have hayfever yet but was trying to get it.
 Right that second I sneezed again, and Pop looked at me and
said, “What’s that grin on your face for?” and I said, “Is there a grin
on my face?”

“There certainly is,” he said, and I sighed and wished I could

sneeze again, which for some reason I did, without even trying to,
or looking at the sun, or smelling the gladiolus or anything, and I got
a quick hope that maybe I was actually going to get hayfever.

Pop banged the car door shut, after taking out a paper bag which
had something in it he’d probably bought somewhere in town. Then
he said, “You’ve maybe been working too hard and been sweating,
and with the wind blowing, you need a dry shirt. Better come in the
house and help your mother and Charlotte Ann and me eat this ice
cream,” which I did, our Sunday school teacher helping also, she
being the reason Pop had hurried to town to get the ice cream in the
first place.

Then Mom told me to go gather the eggs, which I started to do,

and ran ker-smack into something very interesting. I was up in our
haymow looking for old Bentcomb’s nest for her daily egg, which
was always there if she laid one, although sometimes she missed a
day.

“Well, what do you know?” I said to myself when I climbed up

over the alfalfa to her corner. Old Bentcomb was still on the nest and
her pretty bent comb was hanging down over her left eye. She was
sitting there like she owned the whole haymow and who was I to be
intruding?

“Hi, Old Bentcomb!” I said, “How’re you this afternoon? Got your
egg laid yet?”

She didn’t budge, but just squatted down lower with her wings all
spread out covering the whole nest.
 “Where’s your egg?” I said, and reached out my hand toward her,
and “zip-zip-peck,” quick as lightning her sharp bill pecked me on the
hand and wrist. She wouldn’t let me get near her without pecking at
me, and when I tried to lift her off to see if she’d laid an egg today,
she was mad as anything, and complained like she was being
mistreated, and gave a saddish disgruntled string of cluck-cluck-
clucks at me and at the whole world.

I let her stay and scooted down the ladder and ran ker-whizz to
the house, stormed into our back door and said to Mom, “Hey, Mom,
Old Bentcomb wants to ‘set’! What’ll we do—break her up or let her
set?”

“For land’s sakes,” Mom said to me, “don’t knock the world off its
hinges!—What! Old Bentcomb!”

“Actually!” I said, “—up in the haymow!”

“We’ll break her up,” Mom said. “We can’t have her hatching a
nest of chickens up there.”

“Couldn’t we make her a nest down here, out by the grape arbor?
Couldn’t we put her in the new coop Pop and I made?”

“Better break her up,” Mom said, “she’s one of our best laying
hens and if we set her, she’ll be busy all summer raising her family,
and not an egg will we get.”

“But we break her up every year, and she never has a family of
her own,” I said. “I think she’d look awfully proud and pretty
strutting around the barnyard with a whole flock of little white
chickens following her,”—which is one of the prettiest sights a boy
ever sees on a farm—a mother hen with a whole flock of fuzzy-
wuzzy little chickens behind and beside and in front of her, and
running quick whenever she clucks for them to come and they all
gather around her and eat the different things which she finds for
them, such as small bugs, pieces of barnyard food, small grains of
this or that and just plain stuff.

“Well, maybe you’re right,” Mom said, all of a sudden, “let’s set
her. First, let’s get her nest ready and select fifteen of the nicest
leghorn eggs we can find and have them all ready for her; then you
go get her and bring her down.”

“She won’t want to leave her nice warm nest up in the haymow,” I
said to Mom, looking up at her kinda pretty, warmish summer face
under its blue sunbonnet.

“No, she won’t,” Mom said back to me, “But she’ll do it if we work
it right. Hens are very particular about moving from one nest to
another. We’ll maybe have to shut her up in the coop.”

Well, it was one of the most interesting things I liked to do around

the farm. First, we took a nice brand new chicken coop which was
just about as high as halfway between my knees and my belt, then
we scooped a foot-in-diameter roundish hole in the ground close to
our grape arbor, making the hole about only a few inches deep. We
lined it with nice clean straw and then selected fifteen of the
prettiest, cleanest white eggs we could find which had been laid that
very day by the different leghorn other hens on our farm, and which
would probably be what were called “fertile eggs” and would hatch.
Then I ran lickety-sizzle as fast as I could to our barn, scooted up
the ladder into our haymow, and in spite of Old Bentcomb’s being
very angry and not wanting to leave her nest, I got her under one
arm and brought her down the ladder.

In less than a jiffy or two, I was with her up to where Mom and I
were going to coop her up in the coop. I stooped down first and
looked into the dark inside of the coop and there was the prettiest,
nicest most beautiful fifteen eggs you ever saw all side by each. The
coop had a roof on it but no floor, the floor being the ground with
the straw nest in it. I pushed Bentcomb very gently and in a friendly
way up to the hole in the front of the coop, and let her look in at the
nest full of eggs. She had been clucking like everything and whining
and complaining in a saddish sort of voice which meant she wanted
to be a mother of a whole flock of little chickens, but say! She was
mad at me and didn’t want to go in. She kept turning away from the
hole in the coop not even looking at the nice new nest. So I said to
her, “O.K., Old Bentcomb, I’ll take you out and show you what will
happen to you if you don’t sit on those eggs.”

I took her in my two hands, holding her tight so she wouldn’t

squirm loose and get away, and walked with her to our chicken
house and around behind it to where there was a peach tree under
which we had a pen with chickenyard wire all around and on top.
Inside were about nine or a dozen of our best laying hens who had
wanted to set, but whom we decided to “break up” instead of letting
them have their stubborn hen-ways and “set.” There they were, all
shut up by themselves. Some of them were walking around with
their wings all spread out, and clucking like they wanted a bunch of
little chickens to come and crawl under them, and they were cluck-
cluck-clucking in a saddish whining tone of voice. Over in one corner
was a white egg which meant that one of the hens had already
given up wanting to “set” and was behaving herself again like a
good laying hen. And I thought that as soon as we could decide
which one of the hens it was, we’d take her out and let her have her
liberty again.

“See there,” I said to Bentcomb, “look at those lonesome old hens!

They’re clucking around just like you’ve been doing. Every one of
them wanted a family of her own, and not one of them is going to
get it! If you don’t be good, and go in that coop like we want you to,
we’ll have to shut you up in here and leave you for two whole
weeks, which we do to all hens who want to ‘set’ and we won’t let
’em.”

Say, Bentcomb wasn’t interested at all. She absolutely refused to

look, so I took her back again to the coop. “I’m going to give you
one more chance,” I said. “I want you to go in there carefully, not
breaking any of those eggs, and behave yourself.”

Once more I got down on my knees, holding her carefully like she
was a very good friend, which she was, and so she could look in and
see for herself what we wanted her to do.

Well sir, this time she must have decided to be good, ’cause all of
a sudden, she quit struggling and looked in like she’d made up her
mind it might be a good place for her to live for awhile. Without me
doing any pushing, or anything, she very slowly started to creep
inside the opening in the coop, toward the eggs. The next thing I
knew she was on the nest, turning around and scooting herself
down and spreading her wings out and settling down and covering
every one of those fifteen eggs with her wings.

I turned and yelled, “MOM! She’s gone in! She’s going to set!”

“Put the board over the hole for a while,” Mom said, “so she can’t
get out. Let her stay until she feels at home, and then she’ll go back
every time we let her out for exercise and water and food.”

I put the rectangular shaped board over the door of Bentcomb’s

house, and propped it shut with a brick, so she couldn’t get out.

And so we “set” my favorite hen, Old Bentcomb. In just three

weeks there’d be a whole nestful of cheeping chicks and a very
proud mamma hen. I sat down for a minute on the roof of her house
to rest. I was almost overworked, I started to think, when Pop
yelled, “Hey, Bill! Come on out! We’ve got to get the rest of the
chores done!” So I started to the barn to help him do them, still
thinking about the camping trip we’d all been invited to take, and
wondering if I could get to go.

“Don’t you feel well?” Pop asked me when I was moving slowly
around in the barn doing different things.
 “Kinda worn out,” I said, and the dust which I’d been stirring up
with a pitchfork over our corn elevator made me sneeze twice.
“Maybe I’ve got hayfever,” I said.

“That’s that straw dust you’re stirring up there,” Pop answered.

“Stirring up?” I asked, and knew Pop was right. You just couldn’t
fool Pop, I thought.

He stopped what he had been doing which was something or

other way up at the other end of the barn, and called to me, “Next
week, we’ll take you to the doctor and have him give you a test to
see what makes you sneeze so much.”

“Some people sneeze a lot because of the rainy weather making

so many different kinds of flowers and weeds grow so much and
making so much pollen, maybe,” I yelled back in a tired voice.

Pop ignored my educational remark, and sent me up in the

haymow to throw down some alfalfa for our Brindle cow. While I was
up there, I stirred up the dust in the hay and sneezed three or four
times real loud.

Just then Pop called up to me and said, “What’s the matter, Bill?
Are you hurt?”—which made me feel foolish.

The sun was shining in through a crack in the barn and I peeped
out like I nearly always do when I’m up there and looked around at
the different things such as three rows of newly hoed potatoes in the
garden. I could hardly believe my eyes, when I noticed that there
were only three rows I’d hoed. It had seemed like seven.

Then my heart almost jumped into my mouth when I heard voices

downstairs and one of them was Barry Boyland’s laughing voice. He
and Pop were talking, and saying they were glad to see each other. I
stopped in my tracks, and listened for all I was worth, and this is
what I heard, “Well, Barry, we have to do something for him—he’s
getting the hayfever so badly. Maybe the North would be good for
him.”

And then Barry laughed the queerest sounding laugh I’d heard in
a long time and said, “Sure, I understand. It’s the same story
wherever I go—the boys of the Sugar Creek Gang are all sneezing
pretty bad, all except Dragonfly, who is better this year than last—
but his parents said he could go, too.” Then Pop and Barry laughed
long and loud at each other like it was funny or something. But I
didn’t care at all. I was so tickled inside.

Right away a terrible scream of happiness jumped up into my

throat, and if I hadn’t stopped it, I’d have yelled even worse than I
do when I’m yelling for our baseball team.... Oh boy, oh boy—
another trip up North this summer, with all the gang going along!
 4

A I’ve already told you, when we were going to get to go North,

S
we didn’t have any idea we’d run into such an exciting and
dangerous mystery, but when a gang of boys get together on a
camping trip in the wild North, something is pretty nearly always
bound to happen, which it did.

On the way we went through a city which advertised itself as the

Capital of the Paul Bunyan Playground—Paul Bunyan being what is
called a mythical lumberman of the North, and was supposed to
have been terribly big like a giant in the story of Jack and the
Beanstalk, which is a fairy story every boy ought to know—only
instead of Paul Bunyan being a bad giant, he was a good one, and
was always doing kind things for people.

We stopped to get some gas for Barry Boyland’s station wagon—

which is what we were all riding in—right across from a tourist camp
called “Green Gables,” and Little Jim gasped and said, “LOOK! Who
and what is that?”

I looked out at what Little Jim was looking at, and saw what he
saw, and it was a great statue of a man with a beard and mustache,
standing with one hand upraised and the other on a back of a statue
of a great big extra big blue cow which had horns. Poetry spoke up
and said, “That’s Paul and Babe.”

“Paul and Babe Who?” Dragonfly wanted to know, and Poetry,

who, as I’ve told you before, had a lot of books in his library, all of a
sudden reached down into a briefcase he had with him and pulled
out a book and said, “That’s Paul Bunyan and his big blue ox, whose
name is Babe. It was the blue ox whose footprints were so large that
when it walked around they sank deep into the ground, and
everywhere it went it left big holes. Then when it rained, the rain
water filled up the holes and that made all the eleven thousand
great big blue-watered lakes which live in Minnesota.”

Little Jim, who likes fairy stories and legends, grinned and said,
“What made the water blue, then? How come?” You see, nearly all
the water in nearly all the great big hundreds of lakes we’d already
seen on our trip was as blue as the blue on the hair ribbon Circus’
sister wore to school at Sugar Creek.

“What made the lakes blue?” asked Poetry with a question mark in
his voice. He puckered his fat forehead, and said, “Blue—oh that!”
He thumbed his way through the Paul Bunyan book quick, to see if
there was anything in the book to explain it, but there wasn’t. So he
said, “Old Babe, the ox, was blue, you know. One day when he was
out swimming in the headwaters of the Mississippi, the blue began
to come off, and pretty soon the Mississippi, which flows through a
lot of lakes up here, was all blue. The water flowed all around from
lake to lake and pretty soon the lakes’ waters were all blue, too!”

Well, it made as good an untrue story as any of the rest of the

exaggerated ones in the Paul Bunyan book so we added it to the list
and decided to tell it to our folks when we got back to Sugar Creek.

Pretty soon we drove on, right straight down through the pretty
little modern-looking city, where there were lots of people walking
the streets in vacation clothes.

Pretty soon we passed a Tourist Information place, on the right

side of the road where there was a very tall cement water tower,
that was shaped exactly like my pop’s big long six-battery flashlight
back home, being a lot larger at the top. Little Jim squinted his
pretty blue eyes up at it like he was thinking about something. Then
we went on, and Poetry read to us different crazy things the
mythical Paul Bunyan was supposed to have done, such as he had
been such a big baby when he was born that it took six large storks
to carry him to his parents; and Paul’s pet mosquitoes dug the wells
up here where we were; his soup bowl was so large it was like a
lake and the cook had to use a boat to get across it; also his
pancake griddle was so large that they greased it by tying greasy
griddlecakes on the bottoms of some men’s shoes and they skated
around over its surface to grease it for Paul—things like that.

Little Jim surprised us all of a sudden by saying, “Anybody want to

hear how all the people decided to move up into this country and
stay here?—How Paul Bunyan and I working together got them to
come, when nobody wanted to?”

“How?” Dragonfly wanted to know. “What do you mean YOU and

Paul Bunyan worked it? Paul used to live here long before you were
born. You never even saw him!”

“Oh I didn’t, didn’t I?” Little Jim asked and had a very mischievous
grin on his innocent face. “Want to hear the story?”

“Sure,” Poetry and I said, and Dragonfly said, “No.”

Little Jim said, “All right, I won’t—anyway, it’s too important a

story to tell to such a small unappreciative audience.” He sighed like
he was sleepy and curled up with his head on my lap and sighed
again and almost before I knew it he was actually asleep.

It felt good having Little Jim lying with his head in my lap, he
being my almost best friend except Poetry, and also being a really
wonderful little guy and was the best Christian in the whole Sugar
Creek Gang. He was always thinking and saying important things
about the Bible and heaven, and the One who had made the world,
and also about His Son who had come here to this pretty world once
and died on a cross which was made out of a tree, just to save
anybody who would repent of his sins and believe on Him.
 I looked down at that pretty curly head, and thought of Sugar
Creek and my parents and little Charlotte Ann, and was lonesome for
a minute. Then pretty soon I was sleepy myself and the flying tires
of the station wagon sort of sang me to sleep too. Once I half woke
up on account of Little Jim wiggled in my lap and I heard him
mumbling something. I was too sleepy to listen, but it sorta sounded
like he maybe thought he was at home getting ready to crawl into
bed and go to sleep. I kinda bent my ear down a little and listened
close to his perspiring face and say! I heard some of the prettiest
words you ever heard in your life and they were, “Now I lay me
down to sleep, I pray the Lord my soul to keep; if I should die
before I wake, I pray the Lord my soul to take....” Little Jim was
kinda mumbling the words. I’d heard the poem before, in fact my
folks had taught it to me, and when I was littler I’d said it at night
myself. But Little Jim said something else I couldn’t quite make out,
but it sounded like this: “Please also—bless—Little Snow-in-the-face,
and help him to get well...” Then I felt Little Jim’s shoulder relax
against my stomach and I knew he was sound asleep. In another
jiffy I was asleep myself.

When I woke up we were still flying along with Barry at the wheel,
and most of us sitting in lying-down positions, getting a swell
afternoon nap. It was wonderful to ride along that fast, and also
wonderful to see all the things we saw, as the road wound itself
around and around like the winding barefoot-boy paths through the
Sugar Creek woods along Sugar Creek itself. At the town of Pass
Lake, most of us got out, stretched ourselves and bought postcards
at a drug store and sent them to our folks. I sent a card that showed
some men climbing a tree, and some great big fish were at the
bottom looking up like hungry bears look up at boys.

I said to my folks, “Pretty soon we’ll be making camp,” which we

did about a quarter of a mile from the place we’d been the year
before on Santa’s lake front property—Santa, as you know, being the
great big laughing fat man who likes boys almost as much as Old
Man Paddler does.
 “Where’s Mrs. Santa?” Poetry asked that kind person, maybe
remembering the blackberry pie she’d given us, and maybe missing
her very friendly and extra special giggle, which we’d all liked to
hear so well. I had looked forward to seeing her laugh with her eyes
as well as hearing her laugh with her bird-like voice.

Santa, who was sitting in his big white boat which was beached
near where we were making camp, and was helping Tom Till get his
fishing pole and line ready for a fishing trip in the morning, said to
Poetry, “She’s gone to California, but will be back early next week,
before you boys have to go back to Sugar Creek.”

Well, it was almost time for the sun to go down, and we would
have to get busy pitching our tents. Barry called to us from the
station wagon which was parked close by, “Hey, Gang! Let’s get the
tents up.... Hey, you, BILL! POETRY! TOM!”

We all beat it and pretty soon were working like boy scouts, doing
what is called “making camp.” Barry’d picked a site not far from the
lake, and also not too far from a wood pile, so a gang of boys who
were lazy only when there was work to do, wouldn’t have to carry
wood too far. Also he picked a place where there wouldn’t be too
much shade so it wouldn’t be too damp, and yet there would be
sunshine every day if there was any.

“Why don’t we put the tents under this big tree right here?” Circus
asked, and Barry said, looking up at the tree, “See that great big
half dead limb there?”

Dragonfly looked up and saw it, and said, “Sure, what of it?” and
Little Jim spoke up and said, “The wind might blow some night,” and
then turned and ran to where Big Jim was, about fifty feet away and
who, with his jack-knife, was cutting green sticks of different sizes to
help us make what Barry called an outdoor kitchen, which he said
was going to be like the kind the Chippewa Indians used to use.
 All of us were either giving or obeying orders, and in a few jiffies
our tents were up and the outdoor kitchen was nearly finished.

“O.K., you guys—you and Poetry,” Barry ordered Poetry and me,
“roll up a couple of those big round rocks over there, get a couple of
forked sticks, and push them right into the fire.” We already had a
roaring fire going in a place where it was safe to have one. No boy
or anybody ought to start a fire in any forest or woods any time
unless it is in a place where there is supposed to be one, and where
it can’t spread, or a whole forest might get burned up.

“What on earth?” I thought, as Poetry and I grunted a round rock

apiece up as close to the hot fire as we could, and then pushed
them the rest of the way with forked sticks so we wouldn’t get
burned, ourselves.

“Wait and see,” Barry said, and we did, but kept wondering, “Why
on earth?” We got two other rocks also, while the rest of the gang
helped put up the tents and made things ready for our first night’s
sleep. I had a tingling feeling all inside of me, and just knew we
were going to have the most wonderful time of our lives.

It didn’t take us long to get supper over, which we cooked

ourselves on a little two-burner pressure gas stove which Barry had
brought along, he not wanting us to take time to cook in real Indian
style, which we would most of the time.

“Ouch!” different ones of us said to each other and all of a sudden

started slapping around at mosquitoes.

“Here—rub this on,” Barry said, “and be careful not to get any too
near your eyes and lips.” He handed us a couple of bottles of
mosquito lotion and we smeared our bare hands and ankles and
necks and ears and faces with the sickenishly-sweetish-smelling
stuff, and right away it was just like there wasn’t a mosquito in the
world.
 Santa came over and we all sat around the camp fire, with the
pretty sparks and flames playing above the beds of coal and the four
large roundish rocks in the middle of it.

After we’d all listened to Barry tell us an honest-to-goodness Bible

story about something that had happened on Galilee Lake once, we
all took turns telling made-up stories. It was Little Jim who
suggested we all make up Paul Bunyan stories and since it was a
good idea, we decided to try to see who could make up the best
one, and so we started. First, though, Barry told us the thrilling and
very interesting Bible story, which maybe I ought to tell you here
myself, ’cause it was one of the best stories a real red-blooded gang
of boys ever heard.

All of us were in a sort of half circle around the camp fire, on

blankets and also on each other, some of us leaning up against each
other, like right that minute I was against Poetry.... “Get over,” Poetry
said to me. “Don’t crowd so close.”

“I’m trying to get warm,” I said. “It’s cold. I’m using you for a
windbreak.”

And Circus said to Poetry—“You’re a windbreak when it’s cold, and

when it’s hot we lie behind you in the shade,”—Poetry, as you know,
being fat as a small cow. Most of us giggled, except Poetry.

“It happened like this,” Barry began,—and I noticed Little Jim

reach into his vest pocket, and pull out his New Testament to look
up the place where Barry was getting the story from. I did the same,
and so did most of the gang, except Tom Till who had forgotten to
bring his. I looked at him and he swallowed like he was
embarrassed, so I reached out mine to him and he sort of looked on,
although I knew he couldn’t see very well, and wasn’t very good at
reading the Bible anyway. Besides it was more interesting to watch
Barry’s brown face and his one all-gold front tooth sparkling in the
firelight when he talked.
 It was one of my very favorite Bible stories and was about some
fishermen who lived near a great big blue-watered lake that was
thirteen miles long and seven miles wide and had thousands of fish
in it. Two brothers, named Peter and Andrew, were fishing, not with
poles but with nets, and two other brothers whose names were
James and John, whose Pop’s name was Zebedee and whose Mom’s
name was Salome, were using another boat. I was feeling sorry for
the double brothers ’cause they hadn’t caught any fish and I was
wondering what their Moms would say when they got home, when
Barry started in to telling about a big crowd of people coming along
listening to Someone telling wonderful stories and also telling them
about the Father in Heaven and how to live right and things like
that; and the crowd got so close to the Speaker that He might have
been crowded into the water.

He turned and asked Peter to let Him borrow his boat, so He could
get into it and push out from shore a little, and then He could talk to
the crowd and not get trampled on and also the crowd’d be able to
see Him. It was a bright idea, I thought, and wished I had been
there, ’cause if it was wonderful to hear our minister at Sugar Creek
tell about Him in his very interesting sermons, it would have been
even wonderfuller to have been beside that pretty blue-watered lake
that day and listened to the Saviour right while He was talking....

Pretty soon, the Speaker’s sermon was over, Barry said, and then
He, just as if He wanted to pay Peter for being so courteous as to let
Him make a pulpit out of his boat, told Peter to shove the boat into
the deep water and let down the nets for some fish.... Say, Peter
didn’t want to do it, ’cause he had been fishing around there all
night, and hadn’t caught anything, and he might have wondered,
“Why do it again and make a fool of myself?”

“But,” said Barry,—and I could see his all-gold tooth shining as he

talked, and even though he was smiling, his face was very sober—“it
is better to obey the Lord, boys, even if it does seem foolish to the
world for you to do it, than it is to disobey Him. Besides, He has a
right to give us orders, since He is the Son of God....”

He kept on talking, but for a minute, I looked at Circus, who I

noticed had his fists doubled up, and was lying on his stomach and
his elbows, looking up and across the fire at Barry. Also he had his
chin resting on his doubled-up fists and the muscles of his jaw were
working and I knew he was maybe imagining himself to be Peter
and his thoughts were right out in that pretty lake, and he was
seeing the whole thing with his mind’s eye like I was....

When my thoughts got back to Barry again, he was farther along

in the story to where the net was suddenly jammed full of great big
bouncing, swishing, lunging, splashing fish, and Peter and Andrew
had to have help to pull the net in. Also right that second, the big
strong net began to break in places and some of the fish were
getting away, so Peter let out a yell for James and John to make a
dive for their boat—in fact, to bring their boat with them which they
did quick, and both of the boats were so jammed full of fish that
both of them started to sink. That scared Peter, and also all of a
sudden Peter realized that the Man he’d been listening to was more
than a man, but was also the Lord. He all of another sudden realized
what a terrible sinner he was, and he forgot all about the bouncing,
swishing, lunging, splashing fish and dropped down on his knees
and cried out to the Lord, “Go away, Lord, leave me. I’m a sinful
man!”—on account of he was so ashamed of himself for being a
sinful man, he didn’t think he was good enough to be anywhere near
the Lord....

But say, Jesus had done all this on purpose to get Peter to believe
in Him, and He told him not to be afraid any longer, but said, “Fear
not; henceforth thou shalt catch men...”

When Barry said that, Circus’ bright eyes lit up and he interrupted
the story to say, “What’d he mean by that?” Before Barry could
answer, Little Tim Till surprised us all by cutting in and saying across
the crackling fire to Circus, “He meant, ‘Don’t be scared; from now
on you’ll be what our Sugar Creek minister calls a soul winner.’”

Well, it was a wonderful true story, and for some reason I had the
happiest feeling all inside of me. I not only wished all of a quick
sudden that I had been there and had maybe been Peter or Andrew
or one of Mother Salome’s two boys, but I felt also that maybe the
most important thing in the world was to be a soul winner, or a
fisher of men...

Well, the story was done and the sky above the lake toward where
the sun had gone down, reminded me of the reddish, purplish and
also yellowish spread-out feathers of a terribly big fantail pigeon.
 5

I WASsitting there on a small log, looking at the extra beautiful sky,

looking at it over the top of our camp fire, thinking about how the
rays of the sun shooting up looked like a lady’s many colored
unfolded fan or the tail of a fantailed pigeon, when Barry said, “One
of the Sporting Clubs up here is offering a prize for the best original
Paul Bunyan story... Here’s a chance for you boys to stretch your
imaginations a little...”

Pretty soon we were all racking our brains to see if we could think
of something about Paul Bunyan that nobody had ever thought of
before, which Barry might decide was good enough to write about
and send in to the contest... Different ones of us made up different
things, such as: One time Paul Bunyan gave a wintertime party in a
terribly big recreational center in Bemidji, and so many people
answered his invitation and came that there wasn’t any place to
hang their fur coats and other heavy coats, so Paul went out and
blew on his horn and hundreds of great big huge antlered deer came
running in from all directions, and Paul stood them up all around the
outer wall of the building, each one of them facing the center, and
the fancy ladies hung their fur coats and other kinds of different
colored coats on the antlers, using them for what is called
“costumers.” Those deer stood there patiently, without moving, with
their kind eyes watching the skaters.

Everything was going fine, until somebody opened all the doors all
around to let in some fresh air, and then all of a sudden, Old Babe,
the blue ox came in and started lumbering around looking for Paul,
and stamped his hoofs and snorted like a mad bull, and the people
got scared and excited and the women started to screaming, and
that scared the hundreds of deer, and they bolted for the doors in a
terribly mad and wild scramble, and, there being doors all around
that were open, they took all the coats with them...

That was Big Jim’s story, and when he told it, I remembered that
he always got very good grades in English in the Sugar Creek
School.

Dragonfly told his story, and it was that Paul Bunyan got hay fever
so bad and sneezed so hard and so many times in succession that it
blew a whole forest over; Poetry said Paul Bunyan ate so many
blackberry pies and got so fat that when he went in swimming in
Leech Lake and splashed around a lot, so much water splashed out
of the lake for hundreds of miles around that it made a thousand
new lakes so that the ten thousand lakes that Minnesota had at first
were changed to eleven thousand; Circus said the day Paul ate
Poetry’s blackberry pies he had to have toothpicks to pick the seeds
out from between his teeth, so he cut down some Norway pines with
his jack-knife which was seven feet long, and used them for
toothpicks; Dragonfly looked at me and my red hair, with a
mischievous grin in his dragonfly-like eyes, and told another story
real quick which was: Paul’s long hair was so red that when he was
asleep one windy day, the Indians saw it blowing in the wind and
thought it was a forest fire. They threw water all over him, and ever
since then, all red-haired people have been all wet.

Well, that was supposed to be funny, and most everybody around

the camp fire thought it was and laughed hard, but it wasn’t funny,
maybe. For a minute I was almost mad, but decided it would be a
waste of good temper to spoil what the others thought funny;
besides my pop says any boy who wants to get along with people
can’t afford to always be taking offense. I couldn’t think of anything
about Paul Bunyan that would help me get even with Dragonfly, so I
let Little Jim tell his story, and we didn’t have time for mine, on
account of it was time to go to bed.
 I watched Little Jim’s small friendly face in the firelight and in the
light of the afterglow of the sun which had already gone to bed, and
he looked so innocent, that you couldn’t tell whether he was thinking
or not, but it was fun to listen to him, ’cause his mouse-like voice
squeaked out the strangest story, which really sounded good, and it
was: “Well, when Paul and I were up here in this pretty country of
many lakes, we got awful lonesome and wished there were some
people living here. We stayed down where Brainerd is now, and Paul
would carry me around in his vest pocket and tell me stories, and
complain about how lonesome he was...”

Well, it sounded like Little Jim was going to have a real good story,
so I listened and sure enough it was. That little innocent-faced guy
said that Paul Bunyan got so lonesome finally that he took his big
long brown flashlight and some different colored cellophane and
stood the flashlight, which was two hundred feet long, up on the
ground, and built a wooden platform around it right at the place
where the switch was, and every night Little Jim sat on that platform
of the two-hundred-foot-tall flashlight and turned that light on and
off and on and off; and Paul would stand beside the flashlight and
slide different colored pieces of cellophane paper across the top of
the flashlight, and the whole sky was all lit up in many different
colors every night, changing just like the beautiful northern lights do
—(and I thought that maybe the sky above the lake had made little
Jim think about the different colors)—and pretty soon in a week or
so, people from Iowa, Missouri, Tennessee, and all the southern
states, began to come up North to see what they thought were
beautiful northern lights, and they liked the country so well they
decided to stay and build their homes, which they did, and so the
town of Brainerd was founded, and then Paul left his flashlight
standing and the people took the big batteries out and used it for a
water tower, where it still stands in downtown Brainerd.

Well, it was a cute idea, and I wished I could think of something

good, but couldn’t, so we broke up our campfire circle, with Santa
standing and yawning his fat self into a straightened up posture. He
looked straight at Tom Till and said, “How about a spin on the lake,
with my new outboard motor, Tom?”

I remembered that Santa and Mrs. Santa didn’t have any children
of their own, and that last year he had liked Tom so well, and had
also been the one who had showed Tom how to become a Christian.
I knew that Tom’s pop was an infidel, and was hardly ever kind to
him, and Tom was maybe hungry for some grown-up person to like
him, so I felt happy inside that Tom was going to get a fast boat
ride, although I wanted to go along worse than anything.

“You, too, Bill—and Poetry, if you like,” Santa said—“if you can
spare them awhile, Barry. I’ll take the rest of the gang tomorrow.
This new motor needs breaking in, you know.”

Well, it was all right with Barry, and it certainly was all right with
me, so away we four went toward the sandy shore to where Santa’s
big white boat was beached, each one of us taking our life preserver
vests, and putting them on before getting into the boat... Boy oh
boy, that lake looked wonderful, having as many colors as the sky
itself, which meant that a lake got its color from the sky, I thought,
and said to Poetry, “Looks like Old Babe, the Ox, must have changed
his colors like a chameleon and taken a swim out here, while we
were telling stories.”

And Poetry surprised me by yelling, “SWELL, BILL, that’s

wonderful! Hey, you guys back there! Bill’s got a good story!”

Well, it made me feel half proud of myself to have Poetry yell that
to the gang like that, and I liked Poetry a lot for a minute, that being
one of the reasons why I liked him anyway—he was always making
a person feel like he was worth something.

It certainly felt fine to sit in the prow of Santa’s big boat, with Tom
Till and Poetry in the middle and Santa himself in the stern, and go
roaring out across the lake. Boy oh boy, in the afterglow of the
sunset, the lake was pretty, and without much wind was as smooth
as Mom’s mirror in our front room at home. I was wishing Pop and
Mom were there, to see things, but wouldn’t want them to stay on
account of I wanted to have some real exciting adventures to tell
them about when we got home...

Pretty soon, our boat cut a wide circle around the end of a neck of
land, and we went roaring down the other side about fifty or maybe
a hundred feet from shore. It was still a little light on the lake, but
the pine trees on the shore looked darkish and it was getting dark
fast. All the time I was wondering if we could run into any exciting
adventures up here in the North when Poetry said, “Look Bill! right
there’s where our boat upset last year and tossed us out, and right
there’s where I hooked that big Northern Pike.”

I remembered and yelled back to him and said so, and went on
thinking—wishing we’d have some kinda scary excitement as well as
a lot of fun camping.

I watched the widening waves that spread out behind us like a

great V, and felt fine and happy, and for some reason I liked
everybody. Also I was remembering the Bible story Barry had told,
and how Peter was afraid to have the Lord anywhere near him,
because he was a sinner, and I began to feel that God was real close
to all of us and I wasn’t a bit scared of Him ’cause I knew that He
had washed all my sins away, which our Sugar Creek minister and
Little Jim say is what He does to a boy or anybody who will really let
Him—washing them away in His own blood.

Just then Poetry yelled to me, “Penny for your thoughts, Bill!” I
started, and looked at him and said, “Look at that reddish sky, will
you?” and Poetry looked and said, “Kinda pretty, isn’t it?”
 6

W E docked at Santa’s dock, and went into his log cabin with him.
It was cozy inside. First he lit two old-fashioned kerosene lamps,
then ’cause it might get cold pretty soon, we helped him start a fire
in his small wood stove in a corner; Tom pumped a pail of water
from the pitcher pump inside the cabin. Santa even had an icebox
with ice in it, and in another small room, twin beds; and back in a
tiny room away back in the back, there was a bathtub and beside it
a very old-fashioned trunk that for some reason made me think of
Robinson Crusoe and buried treasure. I wished harder than ever that
we would run into a mystery up here in the North.... I was all
tingling inside, wanting one so bad. But, of course, I wouldn’t want
the kind that would half scare a boy half to death, like the ones that
sometimes happened to the Sugar Creek Gang, but I wanted an
ordinary mystery anyway.

Pretty soon it would be time to go back to camp and get to sleep.

I was wondering how we could keep warm in our cold wall tents—
which was the kind ours were—when there wouldn’t be any fires
inside and we didn’t have any heaters. Of course I knew I’d be
pretty warm myself, after I’d crawled into my sleeping bag, which is
a waterproof bed made out of khaki drill. It had a soft kapoc filled
mattress, and I would just crawl into it, zip up the zipper slide
fastener on the side, and there I’d be, but it’d be cold to get
undressed and before getting into my pajamas.

Santa showed us different things in his cottage, such as a large

mounted fish on the wall which Mrs. Santa had caught, and also a
great big bearskin rug which was on the floor and had a fierce bear’s
head with wide open red mouth on one end of it; also there was a
snake skin on the wall, which a missionary in Africa had sent him.
 Well, it was soon time to go home. Poetry looked at Santa’s wood
box and said all of a sudden, “You need a load of wood—better let
Bill carry one in for you.”

“Fine,” I said, “I’ll hold the flashlight for you.” I took a flashlight off
the table, and started toward the door with Poetry right after me.

Outside, we looked back through the window at the pretty little

cabin and at Santa and Tom standing by the fire warming
themselves, and all of a sudden Poetry said, “I wish Tom had a pop
like—I wish Santa was Tom’s daddy.”

I thought of old hook-nosed John Till at Sugar Creek and knew

that maybe right that very minute he was probably standing at the
bar in a beer joint sousing his fat stomach with beer, and that Tom’s
mother was maybe not even going to have enough money to buy
groceries for the family the rest of that week.

At the long wood rick, Poetry and I stopped and he said, “Sh! Turn
off the light. I heard something.” I snapped off the flashlight, peered
out into the dark and listened. “It’s a crazy loon,” I said, when one of
those diving birds away out on the dark lake somewhere had let out
a long-tailed quavering cry, which came echoing across to where we
were. Also right that second another loon, closer to the shore,
answered him.

And then my hair started to stand up on end, ’cause I heard

another sound almost like that of a loon, but it wasn’t coming from
that lake. It sounded like a little girl crying and came from over in
the direction of the boathouse where Santa kept his boat in the
winter and his tools and oars and things in the summer.

Then I heard the sound again, plain as day, a faint cry like a loon
that somebody was trying to smother, and maybe had his fingers on
its throat...
 Poetry’s hand was tightening on my shoulder, and his face was
close to my neck, and I could hear and feel him breathing. “Over
there,” he whispered huskily, “close to the boathouse. Down!” he
hissed, and drew me down beside him, both of us hiding behind the
wood rick.

Before I ducked, though, I’d looked in the direction of the

boathouse which was up against the edge of a steep hill, and I saw
a tiny glow like somebody had drawn on a cigarette or cigar and it
had made it glow in the dark.

I knew it couldn’t be any of our camping party ’cause not a one of

us smoked, not even Barry.

Then we heard the boathouse door creaking on its hinges and I

knew I was beginning to be scared.

“It’s a man smoking,” Poetry hissed in my ear, but I didn’t want to

believe it. “Maybe it was a lightning bug,” I said. There were several
of them flashing their spooky little lamps on and off out near Santa’s
boat.

“Lightning bugs’ lights are a yellowish green,” Poetry said, “and

that was a reddish glow.”

I knew he was right but wished in spite of wanting a mystery that

whatever it was, it wasn’t some criminal. Then I heard what
sounded like a stifled cry again and knew it wasn’t any loon, but said
to Poetry, “It’s a loon’s echo, maybe.”

I had the flashlight in my hand and without thinking, but just

doing what I wanted to, shot its long white beam right straight
toward the boathouse, up against the hill. Before I could even think
Poetry had reached out a hand and grabbed my arm and smothered
the light against his fat side, but not before I saw what I saw, which
was a dark shadow of something dart behind the boathouse.
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