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Methods in
Molecular Biology 1711

Louise von Stechow Editor

Cancer Systems
Biology
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
Cancer Systems Biology

Methods and Protocols

Edited by

Louise von Stechow

NNF Center for Protein Research, University of Copenhagen
Copenhagen, Denmark
Editor
Louise von Stechow
NNF Center for Protein Research
University of Copenhagen
Copenhagen, Denmark

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7492-4 ISBN 978-1-4939-7493-1 (eBook)
https://doi.org/10.1007/978-1-4939-7493-1

Library of Congress Control Number: 2017961108

© Springer Science+Business Media LLC 2018

Chapters 5, 6 and 7 are licensed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/). For further details see license information in the chapters.
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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The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

Cancer is a highly complex disease that is often characterized by vast changes in the genetic
and epigenetic landscape. Those changes result in altered protein expression levels in tumors
compared to healthy tissues. Moreover, posttranscriptional alterations lead to deregulation
of signaling processes, and altered metabolic pathways can produce aberrant metabolic
signatures in cancer cells.
A wealth of high-throughput information has emerged over the last decade, including
global measurements of genes, proteins, and metabolites, as well as many other molecular
species. Those studies provide a glimpse of the molecular makeup of cancer cells on various
levels. In order to classify tumor types and predict clinical outcomes of cancer, researchers
often employ sophisticated computational tools to extract cancer-specific events from the
excessive amounts of data that have been compiled.
This volume on “Cancer Systems Biology” comprises protocols, which describe systems
biology methodologies and computational tools, offering a variety of ways to analyze
different types of high-throughput cancer data. Those include for example network- and
pathway-based analyses. Other chapters cover descriptive and predictive mathematical mod-
els used to analyze complex cancer phenotypes and responses to anticancer drugs.
A number of chapters give an overview of data types available in large-scale data
repositories, describe state-of-the-art computational methods used, and highlight key
trends in the field of cancer systems biology.

Copenhagen, Denmark Louise von Stechow

v
Contents

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

PART I SYSTEMS BIOLOGY OF THE CANCER GENETIC AND EPIGENETIC

LANDSCAPE
1 Detection of Combinatorial Mutational Patterns in Human Cancer
Genomes by Exclusivity Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Hua Tan and Xiaobo Zhou
2 Discovering Altered Regulation and Signaling Through Network-based
Integration of Transcriptomic, Epigenomic, and Proteomic Tumor Data . . . . . . 13
Amanda J. Kedaigle and Ernest Fraenkel
3 Analyzing DNA Methylation Patterns During Tumor Evolution . . . . . . . . . . . . . . 27
Heng Pan and Olivier Elemento
4 MicroRNA Networks in Breast Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Andliena Tahiri, Miriam R. Aure, and Vessela N. Kristensen
5 Identifying Genetic Dependencies in Cancer by Analyzing siRNA
Screens in Tumor Cell Line Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
James Campbell, Colm J. Ryan, and Christopher J. Lord

PART II SYSTEMS ANALYSES OF SIGNALING NETWORKS IN CANCER CELLS

6 Phosphoproteomics-Based Profiling of Kinase Activities
in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Jakob Wirbel, Pedro Cutillas, and Julio Saez-Rodriguez
7 Perseus: A Bioinformatics Platform for Integrative Analysis
of Proteomics Data in Cancer Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Stefka Tyanova and Juergen Cox
8 Quantitative Analysis of Tyrosine Kinase Signaling Across
Differentially Embedded Human Glioblastoma Tumors . . . . . . . . . . . . . . . . . . . . . 149
Hannah Johnson and Forest M. White

PART III SYSTEMS ANALYSIS OF CANCER CELL METABOLISM

9 Prediction of Clinical Endpoints in Breast Cancer Using NMR

Metabolic Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Leslie R. Euceda, Tonje H. Haukaas, Tone F. Bathen,
and Guro F. Giskeødegård

vii
viii Contents

PART IV SYSTEMS BIOLOGY OF METASTASIS AND TUMOR/

MICROENVIRONMENT INTERACTIONS

10 Stochastic and Deterministic Models for the Metastatic Emission Process:

Formalisms and Crosslinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Christophe Gomez and Niklas Hartung
11 Mechanically Coupled Reaction-Diffusion Model to Predict Glioma
Growth: Methodological Details. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
David A. Hormuth II, Stephanie L. Eldridge, Jared A. Weis,
Michael I. Miga, and Thomas E. Yankeelov
12 Profiling Tumor Infiltrating Immune Cells with CIBERSORT . . . . . . . . . . . . . . . 243
Binbin Chen, Michael S. Khodadoust, Chih Long Liu,
Aaron M. Newman, and Ash A. Alizadeh
13 Systems Biology Approaches in Cancer Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Aaron DeWard and Rebecca J. Critchley-Thorne

PART V MODELING DRUG RESPONSES IN CANCER CELLS

14 Bioinformatics Approaches to Predict Drug Responses

from Genomic Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Neel S. Madhukar and Olivier Elemento
15 A Robust Optimization Approach to Cancer Treatment under Toxicity
Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Junfeng Zhu, Hamidreza Badri, and Kevin Leder
16 Modeling of Interactions between Cancer Stem Cells
and their Microenvironment: Predicting Clinical Response. . . . . . . . . . . . . . . . . . . 333
Mary E. Sehl and Max S. Wicha
17 Methods for High-throughput Drug Combination Screening
and Synergy Scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Liye He, Evgeny Kulesskiy, Jani Saarela, Laura Turunen, Krister
Wennerberg, Tero Aittokallio, and Jing Tang

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Contributors

TERO AITTOKALLIO  Institute for Molecular Medicine Finland (FIMM), University of

Helsinki, Helsinki, Finland; Department of Mathematics and Statistics, University of
Turku, Turku, Finland
ASH A. ALIZADEH  Division of Oncology, Department of Medicine, Stanford Cancer
Institute, Stanford University, Stanford, CA, USA; Division of Hematology, Department
of Medicine, Stanford Cancer Institute, Stanford University, Stanford, CA, USA; Stanford
Cancer Institute, Stanford University, Stanford, CA, USA; Institute for Stem Cell Biology
and Regenerative Medicine, Stanford University, Stanford, CA, USA
MIRIAM R. AURE  Department of Cancer Genetics, Institute for Cancer Research, The
Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
HAMIDREZA BADRI  Industrial and Systems Engineering, University of Minnesota,
Minneapolis, MN, USA
TONE F. BATHEN  Department of Circulation and Medical Imaging - MR Center, Faculty of
Medicine and Health Sciences, NTNU - Norwegian University of Science and Technology,
Trondheim, Norway
JAMES CAMPBELL  CRUK-Centre Core Bioinformatics Facility, Department of Data
Science, The Institute of Cancer Research, London, UK
BINBIN CHEN  Department of Genetics, Stanford University School of Medicine, Stanford,
CA, USA
JUERGEN COX  Computational Systems Biochemistry Group, Max-Planck Institute of
Biochemistry, Martinsried, Germany
REBECCA J. CRITCHLEY-THORNE  Cernostics, Inc., Pittsburgh, PA, USA
PEDRO CUTILLAS  Barts Cancer Institute, Queen Mary University of London, London, UK
AARON DEWARD  Cernostics, Inc., Pittsburgh, PA, USA
STEPHANIE L. ELDRIDGE  Institute for Computational and Engineering Sciences, The
University of Texas at Austin, Austin, TX, USA; Biomedical Engineering, The University
of Texas at Austin, Austin, TX, USA
OLIVIER ELEMENTO  Department of Physiology and Biophysics, Institute for Precision
Medicine, Institute for Computational Biomedicine, Weill Cornell Medical College,
New York, NY, USA
LESLIE R. EUCEDA  Department of Circulation and Medical Imaging - MR Center, Faculty
of Medicine and Health Sciences, NTNU - Norwegian University of Science and
Technology, Trondheim, Norway
ERNEST FRAENKEL  Computational and Systems Biology, Massachusetts Institute of
Technology, Cambridge, MA, USA; Department of Biological Engineering, Massachusetts
Institute of Technology, Cambridge, MA, USA
GURO F. GISKEØDEGÅRD  Department of Circulation and Medical Imaging - MR Center,
Faculty of Medicine and Health Sciences, NTNU - Norwegian University of Science and
Technology, Trondheim, Norway; St. Olavs Hospital, Trondheim University Hospital,
Trondheim, Norway
CHRISTOPHE GOMEZ  Aix Marseille Université, CNRS, Centrale Marseille, Marseille,
France

ix
x Contributors

NIKLAS HARTUNG  Department of Clinical Pharmacy and Biochemistry, Freie Universit€ at

Berlin, Berlin, Germany; Institute of Mathematics, Universit€ a t Potsdam, Potsdam,
Germany
TONJE H. HAUKAAS  Department of Circulation and Medical Imaging - MR Center,
Faculty of Medicine and Health Sciences, NTNU - Norwegian University of Science and
Technology, Trondheim, Norway
LIYE HE  Institute for Molecular Medicine Finland (FIMM), University of Helsinki,
Helsinki, Finland
DAVID A. HORMUTH  Institute for Computational and Engineering Sciences, The University
of Texas at Austin, Austin, TX, USA
HANNAH JOHNSON  Department of Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA, USA; Signalling Laboratory, The Babraham Institute,
Cambridge, UK
AMANDA J. KEDAIGLE  Computational and Systems Biology, Massachusetts Institute of
Technology, Cambridge, MA, USA
MICHAEL S. KHODADOUST  Division of Oncology, Department of Medicine, Stanford Cancer
Institute, Stanford University, Stanford, CA, USA; Division of Hematology, Department
of Medicine, Stanford Cancer Institute, Stanford University, Stanford, CA, USA; Stanford
Cancer Institute, Stanford University, Stanford, CA, USA
VESSELA N. KRISTENSEN  Department of Clinical Molecular Biology (EpiGen), Division
of Medicine, Akershus University Hospital, Lørenskog, Norway; Department of Cancer
Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University
Hospital, Oslo, Norway
EVGENY KULESSKIY  Institute for Molecular Medicine Finland (FIMM), University of
Helsinki, Helsinki, Finland
KEVIN LEDER  Industrial and Systems Engineering, University of Minnesota, Minneapolis,
MN, USA
CHIH LONG LIU  Division of Oncology, Department of Medicine, Stanford Cancer Institute,
Stanford University, Stanford, CA, USA
CHRISTOPHER J. LORD  The Breast Cancer Now Toby Robins Breast Cancer Research Centre
and CRUK Gene Function Laboratory, The Institute of Cancer Research, London, UK
NEEL S. MADHUKAR  Department of Physiology and Biophysics, Institute for Precision
Medicine, Institute for Computational Biomedicine, Weill Cornell Medical College,
New York, NY, USA
MICHAEL I. MIGA  Biomedical Engineering, Vanderbilt University, Nashville, TN, USA;
Department of Radiology, Vanderbilt University, Nashville, TN, USA; Department of
Radiological Sciences, Vanderbilt University, Nashville, TN, USA; Diagnostic Medicine,
The University of Texas at Austin, Austin, TX, USA
AARON M. NEWMAN  Division of Oncology, Department of Medicine, Stanford Cancer
Institute, Stanford University, Stanford, CA, USA; Institute for Stem Cell Biology and
Regenerative Medicine, Stanford University, Stanford, CA, USA
HENG PAN  Department of Physiology and Biophysics, Institute for Precision Medicine,
Institute for Computational Biomedicine, Weill Cornell Medical College, New York, NY,
USA
COLM J. RYAN  Systems Biology Ireland, University College Dublin, Dublin 4, Ireland
JANI SAARELA  Institute for Molecular Medicine Finland (FIMM), University of Helsinki,
Helsinki, Finland
 Contributors xi

JULIO SAEZ-RODRIGUEZ  Joint Research Center for Computational Biomedicine

(JRC-COMBINE), Faculty of Medicine, RWTH Aachen University, Aachen, Germany;
European Molecular Biology Laboratory - European Bioinformatics Institute
(EMBL-EBI), Cambridge, UK
MARY E. SEHL  Division of Hematology-Oncology, Department of Medicine, David Geffen
School of Medicine, University of California, Los Angeles, CA, USA; Department of
Biomathematics, David Geffen School of Medicine, University of California, Los Angeles,
CA, USA
ANDLIENA TAHIRI  Department of Clinical Molecular Biology (EpiGen), Division of
Medicine, Akershus University Hospital, Lørenskog, Norway
HUA TAN  Department of Radiology, Wake Forest School of Medicine, Center for
Bioinformatics & Systems Biology, Winston-Salem, NC, USA
JING TANG  Institute for Molecular Medicine Finland (FIMM), University of Helsinki,
Helsinki, Finland; Department of Mathematics and Statistics, University of Turku, Turku,
Finland; Institute of Biomedicine, University of Helsinki, Helsinki, Finland
LAURA TURUNEN  Institute for Molecular Medicine Finland (FIMM), University of
Helsinki, Helsinki, Finland
STEFKA TYANOVA  Computational Systems Biochemistry Group, Max-Planck Institute of
Biochemistry, Martinsried, Germany
JARED A. WEIS  Department of Biomedical Engineering, Wake Forest School of Medicine,
Winston-Salem, NC, USA; Comprehensive Cancer Center, Wake Forest Baptist Medical
Center, Winston-Salem, NC, USA
KRISTER WENNERBERG  Institute for Molecular Medicine Finland (FIMM), University of
Helsinki, Helsinki, Finland
FOREST M. WHITE  Department of Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA, USA; Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA, USA
MAX S. WICHA  Department of Internal Medicine, University of Michigan, Ann Arbor, MI,
USA
JAKOB WIRBEL  Joint Research Center for Computational Biomedicine (JRC-COMBINE),
Faculty of Medicine, RWTH Aachen University, Aachen, Germany; Institute for Pharmacy
and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany
THOMAS E. YANKEELOV  Institute for Computational and Engineering Sciences, The
University of Texas at Austin, Austin, TX, USA; Biomedical Engineering, The University
of Texas at Austin, Austin, TX, USA; Diagnostic Medicine, The University of Texas at
Austin, Austin, TX, USA; Livestrong Cancer Institutes, The University of Texas at Austin,
Austin, TX, USA
XIAOBO ZHOU  Department of Radiology, Wake Forest School of Medicine, Center for
Bioinformatics & Systems Biology, Winston-Salem, NC, USA
JUNFENG ZHU  Industrial and Systems Engineering, University of Minnesota, Minneapolis,
MN, USA
 Part I

Systems Biology of the Cancer Genetic and Epigenetic

Landscape
 Chapter 1

Detection of Combinatorial Mutational Patterns in Human

Cancer Genomes by Exclusivity Analysis
Hua Tan and Xiaobo Zhou

Abstract
Cancer genes may tend to mutate in a co-mutational or mutually exclusive manner in a tumor sample of a
specific cancer, which constitute two known combinatorial mutational patterns for a given gene set.
Previous studies have established that genes functioning in different signaling pathways can mutate in the
same sample, i.e., a tumor from one patient, while genes operating in the same pathway are rarely mutated
in the same cancer genome. Therefore, reliable identification of combinatorial mutational patterns of
candidate cancer genes has important ramifications in inferring signaling network modules in a particular
cancer type. While algorithms for discovering mutated driver pathways based on mutual exclusivity of
mutations in cancer genes have been proposed, a systematic pipeline for identifying both co-mutational and
mutually exclusive patterns with rational significance estimation is still lacking. Here, we describe a reliable
framework with detailed procedures to simultaneously explore both combinatorial mutational patterns
from public cross-sectional gene mutation data.

Key words Cancer genomics, Co-mutation, Mutual exclusivity, Signaling pathway, Hypergeometric test

1 Introduction

Genetic aberrations and deleterious environment exposure orches-

trate to govern the development of various human diseases includ-
ing cancer [1–7]. In particular, somatic driver mutations
accumulating in the human genome are largely recognized as the
culprit of human cancer initiation/progression [1, 2, 4]. While
numerous somatic mutations can be detected in a single tumor,
the mutations are distributed across the genome in a cancer-specific
and sample-specific manner [4, 5, 8, 9]. The cancer-specific prop-
erty refers to the scenario that mutational pattern varies between
different cancer tissue types, e.g., liver, lung and breast cancers;
while the sample-specific sense corresponds to the mutational vari-
ety between different patient samples with the same cancer type.
For a specified cancer type, some genes are altered commonly across
patient samples, while others exemplify apparent sample-specificity

Louise von Stechow (ed.), Cancer Systems Biology: Methods and Protocols, Methods in Molecular Biology, vol. 1711,
https://doi.org/10.1007/978-1-4939-7493-1_1, © Springer Science+Business Media, LLC 2018

3
4 Hua Tan and Xiaobo Zhou

Tumor Samples

Gene 1 Co-mutational
Gene 2 pattern

Gene 1 Mutually exclusive

Gene 2 pattern

Mutant Wild type

Fig. 1 Schematic representation of two combinatorial mutational patterns studied in this protocol: the
co-mutational pattern (upper panel) refers to the scenario that a set of genes tends to mutate simultaneously
in a tumor sample, whereas the mutually exclusive pattern (lower panel) represents the opposite scenario:
genes in a given set tend to avoid mutating simultaneously in any one tumor sample

[5]. Previous experimental and statistical analyses have consistently

revealed two combinatorial mutational patterns for a given set of
genes, termed co-mutational and mutually exclusive patterns
[5, 8–10]. As shown in Fig. 1, the co-mutational pattern occurs
when a set of genes tend to mutate simultaneously in a single
tumor, while the mutually exclusive pattern refers to the scenario
in which one and only one of a set of genes is likely to be altered in a
tumor.
Mutually exclusive genes are likely to function in the same
signaling pathway, whereas co-mutational genes are likely to take
effect in different pathways [11]. Combinatorial patterns of genes
can be leveraged to infer signaling networks implicated in human
cancer development and progression. Indeed, many efforts have
been devoted to de novo discover novel driver pathways based on
mutual exclusivity of gene mutations [11–13]. Therefore, it has
essential biological relevance to identify gene pairs or gene sets with
significant combinatorial mutational patterns.
Previous work proposed a statistical method to deal with this
question and nominated a number of gene sets with significant
combinatorial patterns [10]. However, this analysis was performed
on a batch of very limited cell line data. The analysis thus lacks an
elaborate procedure to preprocess data from a giant mutation
database which consists of a large number of clinical samples of
various cancer types (e.g., the recently released Catalog of Somatic
Mutations in Cancer—COSMIC [14] and the Cancer Genome
Atlas—TCGA, https://tcga-data.nci.nih.gov/tcga/). In addition,
the analysis by Yeang et al. adopted different hypothesis tests to
estimate the significance levels of the two combinatorial mutational
patterns, which tend to yield a too conservative p-value for the
co-mutational pattern [10].
To address these issues, we here describe a systematic and reliable
pipeline to identify both combinatorial mutational patterns in cancer
genomes. Here, somatic mutations exclude the synonymous point
 Combinatorial Mutational Patterns in Human Cancers 5

mutations which will not change an amino acid (marked as “coding

silent” in COSMIC). Those mutations typically have little, if any,
impact on the biological function of corresponding proteins and are
uninformative for signaling pathway inference [11, 15]. Other types
of mutations, such as missense and nonsense point mutations, small
insertions and deletions, frame shifts, gene fusions, and transloca-
tions, etc., could be counted as effective mutations when performing
exclusivity analysis. Furthermore, the mutations should be detected
based on genome-wide or exome-wide screening efforts ensuring
that all protein-coding genes were covered, to minimize the statisti-
cal bias induced by incomplete sample coverage.
The step-by-step procedure for data acquisition and criteria of
data quality control, as well as the specific formulae used to calcu-
late the likelihood ratio (LR) and significance level ( p-value), are
elucidated in the following sections. Figure 2 illustrates the overall
procedures of this pipeline for mutational pattern determination.

Mutaton
records in
database

Quality control

Preprocessing

Calculate Calculate
likelihood rato signifcance
level

Determine
mutatonal paern
For Inferring
signaling
network

Fig. 2 Schematic of the overall pipeline proposed in this protocol. The specific
steps of text processing, computation, and visualization are provided in
Subheading 3
6 Hua Tan and Xiaobo Zhou

This pipeline has been shown to be highly effective and efficient in

identifying mutational patterns of gene pairs in cancer mutation
data from COSMIC v68, as described in our earlier publication
[5]. We recently applied this pipeline to analyze the data from the
latest COSMIC release (version 76), which has been threefold
expanded since the release of v68, and well recapitulated and sig-
nificantly improved the previous results (data not shown). How-
ever, our previous efforts were mainly devoted to the biological
discoveries instead of technical details of the analysis. In this proto-
col, we address this gap by providing extensive practical details and
highlighting alterative solutions when encountering problems in
the users’ particular applications.

2 Materials

The pipeline proposed in this protocol has been successfully tested

in the Catalog of Somatic Mutations in Cancer (COSMIC, release
v68 and v76). Therefore, the procedures described in the below
section will be mainly based on the COSMIC database. However, it
is noteworthy that this protocol is applicable to other databases
such as TCGA that contain information of both gene mutation and
associated patient sample IDs. Synonymous mutations and muta-
tions that are not from a genome-wide or exome-wide study should
be excluded prior to further analysis. All the text processing,
subsequent computation, and visualization can be implemented in
Matlab (The Math Works, Inc.), as described previously [5], or in R
[16], another popular language for data analysis and graphics.

3 Methods

3.1 Data Quality 1. Extract mutations of a designated cancer type from the mixed
Control mutation records in COSMIC by the keyword “Primary site”
and Preprocessing (see Note 1).
of COSMIC Mutation 2. Remove synonymous mutations by the keyword “Substitution-
Entries coding silent” (see Note 1).
3. Remove mutation records that are not from a genome-wide
study by the keyword “genome-wide screen” (see Note 1).
4. Generate a gene mutation pattern matrix based on the muta-
tions and sample IDs. The rows and columns of the matrix refer
to samples and genes, respectively. The entry at row i and
column j of the matrix refers to the number of mutations
occurring on gene j in tumor sample i. Figure 3 highlights an
example showing the 9th tumor sample has a mutation on gene
2 by marking the coordinate (9,2) (see Note 2).
 Combinatorial Mutational Patterns in Human Cancers 7

Genes Genes Genes

A 1 2 3 4 5 6 7 8 9 10
B 1 2 4 7 8 9 10
C 1 2 4 7 8 9 10
12 11 10 9 8 7 6 5 4 3 2 1

12 11 10 9 8 7 6 5 4 3 2 1

12 10 9 8 7 6 5 4 3 2 1
Samples

(9,2) (9,2) (9,2)

Mutant Wild type

Fig. 3 Schematic depicting the mutation pattern matrix and entry filtering criteria. (a) A mutation pattern matrix
is generated to represent the mutation profiles of the tumor samples across all genes. A gray grid indicates the
corresponding sample has at least one mutation on the gene specified by the column ID. (b) Columns 3, 5, and
6 are deleted since the associated genes are mutated in only a small fraction of samples (the threshold of
fraction can be prescribed). (c) Row 11 is deleted as the corresponding sample has no mutation in the
remaining genes after the processing in (b)

3.2 Calculation 1. Exclude genes (columns) that do not exceed a prescribed

of Likelihood threshold of sample coverage. As shown in Fig. 3a, b, if the
of Co-occurrence percentage of nonzero entries in a column is lower than a
of Mutant Genes threshold, then delete this column (see Note 3).
2. Remove samples (rows) that do not harbor any mutations
across the remaining genes, as shown in Fig. 3b, c.
3. Calculate the likelihood ratio LRcomb of co-occurrence for each
gene pair by the formula (1):

P ðg1 ¼ 1; g2 ¼ 1Þ
LR comb ¼ ð1Þ
P ðg1 ¼ 1ÞP ðg2 ¼ 1Þ

where P(g1 ¼ 1) and P(g1 ¼ 1, g2 ¼ 1) correspond to the

percentage of samples in which a single gene or both the genes
are mutated, respectively.

4. Determine the threshold of the likelihood ratio (LRcomb) for

pattern categorization based on a mixture Gaussian distribu-
tion fitting model using an Expectation-Maximization algo-
rithm [17]. Specifically, suppose m1, m2 are the means of the
low and high components of all LRcomb’s, and δ1, δ2 their
standard deviations respectively. Then the thresholds for the
co-mutational pattern (lower bound) and exclusive pattern
(upper bound) are calculated as θ1 ¼ m2  δ2/2 and
θ2 ¼ m1 + δ1/2, respectively (see Note 4).
8 Hua Tan and Xiaobo Zhou

3.3 Calculation 1. Calculate the significance level of the co-mutational pattern Pco
of Significance by the hypergeometric test as the formula (2):
of Combinatorial
n2 
X    
Mutational Patterns n1 n  n1 n
P co ¼ = ð2Þ
k n2  k n2
k¼n12

where n, n1, n2, n12 represent the numbers of total samples,

samples harboring gene 1 mutation, samples harboring gene
2 mutation, and samples harboring both gene 1 and gene
2 mutations, respectively.

2. Calculate the significance level of the mutually exclusive pattern

Pexcl by formula (3):

n12 
X    
n1 n  n1 n
P excl ¼ = ð3Þ
k n2  k n2
k¼0

where n, n1, n2, n12 are defined as in the formula of Pco above.

4 Interpretation of the Results

Both co-mutational and mutually exclusive patterns of gene pairs

have biological meaning implicated in signaling network inference.
For the co-mutational pattern, genes are likely to function in
different signaling pathways and exert synergistic impact on
tumor progression. Therefore, multiple oncogenic pathways
driving the tumorigenesis for a particular cancer type could be
identified by analyzing these co-mutational patterns. For the exclu-
sive pattern, more insights could be obtained. In particular, affilia-
tion of genes to a signaling pathway can be inferred from a list of
gene pairs with exclusive pattern. For example, if A-B, B-C, and
C-A are all exclusive gene pairs, then it is reasonable to conclude
that genes A, B, and C are likely to operate in the same signaling
pathway for the particular cancer type in question. When the whole
list of gene pairs is visualized in one graph, as shown in the bottom
of Fig. 2 (by Cytoscape [18]), a signaling network can emerge.
However, this preliminary signaling network subjects to modifica-
tion based on prior knowledge about gene-gene/protein-protein
interactions and experimental evidence for real applications (see
Note 5). To conclude, the exclusive patterns can be used to infer
a specific cancer-associated signaling pathway, while the
co-mutational patterns can assist in exploring whether multiple
oncogenic pathways were involved, as described in our previous
work [5] and references therein.
 Combinatorial Mutational Patterns in Human Cancers 9

5 Notes

1. If combinatorial mutational pattern analysis needs to be con-

ducted on tumor subtypes instead of tissue types, the keyword
“Site subtype” can be used to further divide mutations into
smaller groups of subtypes. For TCGA data, since genomic and
epigenomic data are deposited separately for different cancer
types, the mutation data for a cancer type of interest can be
downloaded directly from the TCGA web site by choosing level
2 MAF (Mutation Annotation Format) file. Also, the informa-
tion of amino acid change corresponding to nucleotide alter-
ation is not available in TCGA, step 2 (Subheading 3.1, which
aims at removing the coding silent mutations) can be skipped.
Since all the mutation data in TCGA are based on exome-wide
sequencing, therefore, it is no necessary to implement the
screening procedure specified in step 3 (Subheading 3.1).
2. When generating the gene mutation pattern matrix, the muta-
tions counted for each gene could be restricted to particular
mutation types such as missense point mutations or gene
fusions, depending on the biological question to be answered
and the working model hypothesis to be tested.
3. The threshold of sample coverage used to exclude the less
frequently mutated genes can be adjusted according to the
sample size and/or gene set size, to yield a reasonable number
of combinations of genes. In our previous practices, the thresh-
old sample coverage was set to 2–10%.
4. When determining the thresholds for mutational pattern cate-
gorization, the mixture Gaussian model based on the
Expectation-Maximization algorithm sometimes can produce
inconsistent outcomes over technical replicates. This is largely
due to the stochastic properties implicated in the EM (Expec-
tation Maximization) optimization procedure. A reliable alter-
native is to simply use LRcomb ¼ 1 to divide candidate gene
pairs into two groups, with LRcomb < 1 referring to exclusive
pattern and the remainder co-mutational pattern. Then rank
the pairs in each group according to P values. After that, select a
reasonable number (e.g., 20–30) of the top-ranked significant
gene pairs in respective groups.
5. Although the pipeline is applied to gene pairs, sets of genes
with particular combinatorial patterns could emerge by inte-
grating gene pairs of corresponding patterns. Thus, the pipe-
line introduced in this protocol can serve as a starting point for
inferring signaling network modules in particular cancers.
10 Hua Tan and Xiaobo Zhou

Acknowledgments

This work was partially supported by the Beijing Normal University

youth funding (105502GK and 2013YB43 to H.T.) and National
Institutes of Health (1U01CA166886, 1R01LM010185, and
1U01HL111560 to X.Z.).

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 Chapter 2

Discovering Altered Regulation and Signaling Through

Network-based Integration of Transcriptomic, Epigenomic,
and Proteomic Tumor Data
Amanda J. Kedaigle and Ernest Fraenkel

Abstract
With the extraordinary rise in available biological data, biologists and clinicians need unbiased tools for data
integration in order to reach accurate, succinct conclusions. Network biology provides one such method for
high-throughput data integration, but comes with its own set of algorithmic problems and needed
expertise. We provide a step-by-step guide for using Omics Integrator, a software package designed for
the integration of transcriptomic, epigenomic, and proteomic data. Omics Integrator can be found at
http://fraenkel.mit.edu/omicsintegrator.

Key words Data integration, Network biology, Computational biology, High-throughput data

1 Introduction

As biologists gain access to increasing amounts of data, the chal-

lenges associated with interpreting those data have increased. Biol-
ogists and clinicians can obtain high-throughput information about
a cell’s genome, transcriptome, epigenome, and proteome with
reasonable effort and constantly decreasing costs. Indeed, much
of those data are freely available to scientists through resources such
as The Cancer Genome Atlas [1] and ENCODE [2]. The challenge
remains, however, in knowing how to interpret those rich datasets.
These “omic” data can be extraordinarily valuable. However, this
value can only be extracted if data are properly analyzed using
methods that account for the relatively high error rate of high-
throughput experiments [3], and then condensed into understand-
able and actionable hypotheses about the underlying biology. This
process can be especially difficult, and especially rewarding, when
attempting to integrate several kinds of high-throughput data. Our
group and others have shown that integrating data from several

Louise von Stechow (ed.), Cancer Systems Biology: Methods and Protocols, Methods in Molecular Biology, vol. 1711,
https://doi.org/10.1007/978-1-4939-7493-1_2, © Springer Science+Business Media, LLC 2018

13
14 Amanda J. Kedaigle and Ernest Fraenkel

sources can lead to novel discoveries that each assay could have
missed on its own [4–6].
Network biology is a fast-growing category of methods for this
type of analysis [7]. Network models provide a valuable resource for
biologists looking to analyze their high-throughput data in a sys-
tems context. By mapping “hits” from high-throughput assays
onto interaction networks, the mechanistic connections between
the hits become obvious, and investigators can focus on pathways,
or series of interactions in the cell that are related to a certain
function, that may be perturbed in the system.
Network methods typically involve modeling the molecules
within a cell—which can for example be DNA, mRNAs, proteins,
or metabolites—as nodes in a graph. Edges between these nodes
connect molecules that are functionally or physically connected
[7]. For example, a protein-protein interaction network (PPI)
would represent the binding of protein A to protein B by drawing
an edge between the “A” node and “B” node in the network.
Several publicly available databases have been created to translate
experimentally discovered protein interactions into PPIs, such as
iRefIndex [8], BioGRID [9], and STRING [10]. There are also
databases that store interactions of proteins with other molecules,
such as metabolites [11–13]. In other types of networks, the edges
can represent more abstract relationships. For example, in a
correlation-based network, edges between nodes might represent
probable co-regulation, rather than physical interactions, based on
covariance between the concentration of molecule A and molecule
B [14, 15].
Mapping high-throughput hits onto networks in search of
affected pathways has several advantages. Hits that are close to
each other in a network might function in the same pathway.
Focusing on subnetworks of functionally related nodes can produce
a more tractable number of targets, rather than the potentially
hundreds of individual factors identified in high-throughput
experiments. In addition, this type of pathway identification
reduces the chance of devoting resources to the analysis of false
positives from the high-throughput screen. Although the confi-
dence for each hit in a screen may be low, the confidence in a
pathway that contains many hits is much higher. Finally, pathway
analysis can help to find novel nodes that may not have appeared in
a high-throughput screen. These “hidden nodes” can be false
negatives in a screen, or true negatives that are nonetheless impor-
tant players in the investigated biological system. Our work has
shown that these hidden nodes can often be important to a system
under study, despite the lack of direct experimental evidence [4, 16,
17]. Using the PPI to discover these pathways de novo, rather than
relying on predetermined pathway databases like KEGG [18],
expands our ability to find novel information, and avoids biasing
the results toward well-studied pathways.
 Network-based Integration of ‘Omic’ Data 15

However, network analysis is not as simple as just mapping

high-throughput assay hits onto PPIs and finding all possible con-
nections through them. Because of the large and highly connected
nature of most biological networks [7], this “brute force” method
results in extremely dense, uninterpretable “hairballs” rather than
clear pathways [16]. Moreover, combining several types of experi-
mental assays into a unified analysis can be complex. For example,
experiments assessing changes in mRNA levels and protein levels
are often not well correlated [19]. It is not trivial to map them onto
one protein or RNA interaction network together. This chapter will
walk you through the use of Omics Integrator, a software package
that proposes a solution to these problems [17].
Omics Integrator is a new software tool designed to help
biologists analyze and synthesize several kinds of high-throughput
omics data, and reduce it to a few important, high-confidence
pathways. Omics Integrator is designed for ease of use by biologists
with basic computer skills (comfort with using the Unix command
line is helpful). Omics Integrator first uses transcriptomic and
epigenomic data to reconstruct transcriptional regulatory net-
works, and then integrates those with proteomic data by mapping
them onto a protein interaction network [17]. It uses two mod-
ules—Garnet and Forest, which are designed to run sequentially,
but can also be run individually. Garnet mines transcriptomic and
epigenomic information in order to predict transcription factors
that may be responsible for gene expression changes in the studied
system. Forest maps these transcription factors and protein-level
experimental information onto a PPI. Forest then implements the
Prize-Collecting Steiner Forest algorithm [16] to predict high-
confidence low-density protein interaction pathways that are
important to the studied system (see Fig. 1).

2 Materials

2.1 Finding 1. Transcriptomics data, i.e., differential gene expression between

Transcriptional different conditions in your study (i.e., tumor vs. control).
Regulators with Garnet 2. Epigenomic data from a source such as TCGA [1], ENCODE
[2], Roadmap [20], Omics Integrator example data, or experi-
mentally derived epigenomic data (in a BED formatted file).
3. Transcription factor sequence binding motif predictions, from
a source such as TRANSFAC [21], and/or Neph et al.
[22]. Omics Integrator provides a file derived from the
TRANSFAC database.

2.2 Network 1. Prize-collecting Steiner tree algorithm executable (msgsteiner

Integration can be downloaded from http://areeweb.polito.it/ricerca/
with Forrest cmp/code/bpsteiner).
16 Amanda J. Kedaigle and Ernest Fraenkel

Fig. 1 Outline of the Omics Integrator workflow. Epigenomic data (open chromatin regions or histone marks)
and transcriptomic data are used to predict influential transcription factors (TFs). Transcription factors and
proteomic data are then mapped onto an interactome, and the Prize Collecting Steiner Forest algorithm is used
to produce small pathways and subnetworks predicted to be relevant to the experimental system

2. Interactome file indicating all known interactions between

proteins. Omics Integrator provides an interactome for
mouse and human proteins derived from iRefIndex [8].
3. Input prize file, indicating the proteins you would like to
include in the final solution (see Note 1).
4. (Optional) Output from Garnet to include transcription factors
implicated by transcriptomic data in the final solution.
5. Cytoscape [25] to visualize the final network solution.

3 Methods

3.1 Installation 1. You can run Omics Integrator as a web tool on our website:
of Omics Integrator http://fraenkel.mit.edu/omicsintegrator/or install it on your
own computer using the instructions at https://github.com/
fraenkel-lab/OmicsIntegrator. You should make sure you have
all dependencies (see Note 2) installed and that you have the
most updated version of Omics Integrator from our GitHub
page (see Note 3).
 Network-based Integration of ‘Omic’ Data 17

3.2 Finding Garnet uses differentially expressed genes from your transcriptomic
Transcriptional assays (i.e., RNA-seq) to predict transcription factors (TFs) that are
Regulators with Garnet likely to be responsible for the altered gene expression. It uses
epigenomic data to find regions of the genome to look for differ-
ential TF binding. For example, this could be ATAC-seq data that
points out accessible regions of the genome in your cell type. The
algorithm will search for transcription factor binding motifs within
regions implicated by your epigenomic data. The strength of these
motifs is then correlated with the magnitude of change of nearby
differentially expressed genes to give each TF a score.
1. Obtain epigenomic data for cell lines related to your samples
from one of the sources listed under Subheading 2.1. Alterna-
tively, if you have epigenomic data for your own samples, you
can use this as well. These data can be in the form of histone
marks ChIP-seq, or DNase-seq or ATAC-seq, all of which
indicate accessible chromatin regions where a TF might be
bound. Collect these data in a BED-formatted file.
2. Go to the Galaxy webserver [23] (see Note 4) to extract the
DNA sequences for your epigenomic regions. Upload your
BED file to Galaxy under the “Get Data” tool, specify which
genome you are using, and then use the “Fetch Alignments/
Sequences”>“Extract Genomic DNA” tool to download a
FASTA-formatted file.
3. Format your experimentally derived gene expression data in a
tab-delimited file with two columns. The first should be the
name of the gene, and the second should be the log-fold-
change of that gene in the study conditions (i.e., tumor
vs. control). We recommend only including genes with a statis-
tically significant change in expression (see Note 5).
4. Create the Garnet configuration file. For an example configu-
ration file, see the README on the Omics Integrator GitHub
page, or the comment on the top of scripts/garnet.py. Your
configuration file should be formatted similarly, but you should
replace the paths to the bedfile, fastafile, and expressionFile with
the paths to the files you created in steps 1–3 in Subheading
3.2. Make sure the annotation files referenced by genefile,
xreffile, and genome are using the correct genome for your
sample (files for mm9 and hg19 are provided with Omics
Integrator).
5. You can change the parameters to your liking (Table 1).
6. Run Garnet on the command line by navigating to the direc-
tory with garnet.py and running python garnet.py yourconfigfile.
cfg. You can also add a --outdir directoryname flag if you would
like to put the output from garnet into a different directory.
Another random document with
no related content on Scribd:
properly prepared gesso is generally laid on the parchment where gold is to come,
so that the raised surface may give the polished metal more effect. The gold over
the bolus was always burnished. It may be noticed that our word ‘size’ is really
‘assise,’ the bed or layer under gilding, for which a gluey substance was suitable.
237. A ‘mordant’ as the word implies is some corrosive liquid, such as is used
by dyers to bite into the fabric and carry in with it the colouring matter. The word
is also employed, as in this passage, for a glutinous size used as ground for gilding,
such as the modern decorator’s ‘gold-size.’ Gold laid in this way has a ‘mat’ surface.
238. The scudo was worth in Tuscany about four-and-sixpence of our money.
In Florence its value was a little greater.
239. See Note 1, ante, p. 248.
240. For the various processes of preparing a panel for painting and for
gilding reference must be made to Cennini’s Trattato, where many technical
matters are elucidated that Vasari passes over almost without notice. It must be
remembered that Cennini writes as a tempera painter, while in Vasari’s time these
elaborate processes were falling out of use. In his chapters 115–119, Cennini gives
recipes for what he calls ‘gesso grosso’ and ‘gesso sottile.’ They are made of the
same materials, ‘volterrano,’ or plaster from Volterra, which is a sulphate of lime
corresponding to our ‘plaster of Paris,’ and size made from parchment shreds; but
the plaster for ‘gesso sottile’ is more finely prepared. The plaster, produced by
calcining gypsum, is first thoroughly slaked by being drenched with water till it
loses all tendency to ‘set,’ and is then as a powder or paste mixed with the heated
size. The size makes the composition dry quite hard, and Cennini speaks of its
having a surface like ivory.
241. See Note 2, ante, p. 248.
242. This we should call ‘shell gold.’ It is in common use. The employment of
the shell represents a very ancient tradition, for shells were the usual receptacles
for pigments in late classical and Early Christian times.
243. This is excellent advice. The architectural character of mosaic decoration,
the distance of the work from the eye, the nature of the technique and material, all
invite to a broad and simple treatment, such as we find in the best mosaics at
Ravenna and Rome. Modern work is often too elaborate and too minute in detail.
244. A modern would say that if the work be really inlaid, it should look like
inlaid work, and not like something else. In the Italy of Vasari’s day however, as we
have seen, painting had so thoroughly got the upper hand, that to ape the nobler
art would seem a legitimate ambition for the mosaicist.
245. The durability of mosaic depends on the cement in which the cubes are
embedded and on the care taken in their setting. The pieces themselves are
indestructible but they will sometimes drop out from the wall. Hence extensive
restorations have been carried out on the Early Christian mosaics at Ravenna and
other places.
 246. In his Proemio delle Vite (Opere, I, 242) Vasari explains what he means
by the words ‘antique’ and ‘old.’ The former refers to the so-called ‘classical’ epoch
before Constantine; the latter to the Early Christian and early mediaeval period,
prior to the Italian revival of the thirteenth century.
247. At S. Costanza (see Note 5, ante, p. 27) on the vault of the aisle there are
decorative mosaics of the time of Constantine showing vine scrolls issuing out of
vases, and classical genii gathering the grapes. Birds are introduced among the
tendrils.
248. The mosaics at Ravenna and S. Marco, Venice, are well known. In the
Duomo at Pisa, in the apse, there still remains the Saviour in Glory between the
Madonna and John the Baptist, designed by a certain Cimabue, and the only
existing work which modern criticism would accept as from the hand of the
traditional father of Florentine painting. It may however have been another painter
nicknamed ‘Cimabue,’ who worked at Pisa early in the fourteenth century. The
mosaics of the Tribune of the Baptistry at Florence were executed in 1225 by
Jacobus, a monk of the Franciscan Order, and this fact is attested by an inscription
in mosaic which forms part of the work.
249. This mosaic, called the ‘Navicella,’ represents the Gospel ship manned by
Christ and the disciples, with Peter struggling in the waves. It has been so much
restored that little if any of Giotto’s work remains in it. It was replaced in the
seventeenth century, after some wanderings, in the porch of the present Basilica,
but Vasari saw it of course in the porch of the old, or Constantinian, church, the
entrance end of which was still standing in his day.
250. This mosaic was executed at the end of the fifteenth century by Domenico
Ghirlandajo and his brother over the northern door of the nave of the cathedral of
Florence. It is still in situ but has been greatly restored. The date 1490 is
introduced in the composition.
251. This corresponds with modern practice. The following is from a paper by
Mr James C. Powell, who, as practical worker in glass, has been engaged with Sir
W. B. Richmond in the decoration in mosaic of the vaults of St Paul’s. ‘The glass
which is rendered opaque by the addition of oxide of tin, is coloured as required by
one of the metallic oxides; this is melted in crucibles placed in the furnace, and
when sufficiently fused is ladled out in small quantities on to a metal table, and
pressed into circular cakes about eight inches in diameter and from three-eighths
to half an inch in thickness; these are then cooled gradually in a kiln, and when
cold are ready for cracking up into tesserae, which can be further subdivided as the
mosaicist requires. It is the fractured surface that is used in mosaic generally, as
that has a pleasanter surface and a greater richness of colour; the thickness of the
cake, therefore, regulates the limit of the size of the tesserae, and the fractured
surface gives that roughness of texture which is so valuable from an artistic point
of view.’ (Transactions, R.I.B.A., 1893–4, p. 249).
 252. This is a point attended to by the best modern workers in mosaic. Where
gold backgrounds are used it is advisable to carry the gold into the figures by using
it as Vasari suggests for the lights on the draperies. If this were not done the figures
would be liable to tell as dull masses against the more brilliant ground. The use of
gold backgrounds is specially Byzantine. The earlier mosaics at Rome and at
Ravenna have backgrounds of blue generally of a dark shade, which is particularly
fine at Ss. Cosma e Damiano at Rome and in the tomb of Galla Placidia at Ravenna.
The mosaics at S. Sophia at Constantinople of the sixth century had gold
backgrounds, and this is the case also with all the later examples in Italy from the
ninth and tenth centuries onwards. The finest displays of these varied fields of
gold, now deep now lustrous of hue, are to be seen in S. Sophia, S. Marco at Venice,
and the Cappella Palatina at Palermo.
Vasari’s account of the fabrication of the gilded tesserae required for this part
of the work is quite clear and agrees with modern practice. The gold leaf is
hermetically sealed between two sheets of glass by the fusion of a thin film over it.
The technique of the ‘fondi d’ oro,’ or glass vessels adorned with designs in gold,
found in the Roman catacombs, was of the same nature.
253. It has been noticed at some places, as at Torcello, that before the cubes
were laid in the soft cement the whole design was washed in in colour on the
surface of the cement. This facilitated correct setting and avoided any appearance
of white cement squeezed up in the interstices between the cubes. On this
particular feature of the mosaic technique Berger has founded an ingenious theory
of the origin of painting in fresco. It is his thesis, in his Beiträge zur Entwicklungs-
Geschichte der Maltechnik, I, München, 1904, that the ancients did not employ the
fresco process, but that this was evolved in early mediaeval days out of the mosaic
technique as seen, e.g., at Torcello. The stucco, that Vasari describes, must be put
on portion by portion, for it only keeps soft two or three days, and can only be used
for setting the cubes while in a moist state. Now, Berger contends, if the design for
the mosaic be painted in colours on the wet stucco, and the whole allowed to dry,
without any use of the mosaic cubes, we should have a painting in fresco, and he
imagines that fresco painting began in this way. Unfortunately for the theory, (1),
the testimony of Vitruvius and Pliny is absolutely decisive in favour of the
knowledge in antiquity of the fresco technique, and, (2), the use of the coloured
painting on the stucco as a guide for the setting of the cubes was not normal, and
can never have been used so freely as to give rise to a new technique of painting. As
a fact, this colouring of the stucco is objected to by the best modern workers on
aesthetic grounds, for they point out that the lines of grey cement between the
coloured cubes answer to the lead lines in the stained glass window, and should be
reckoned with by the designer as part of his artistic effect. No doubt the older
mosaicists, like the workers in stained glass, instinctively apprehended this, and
had no desire for the coloured cement.
254. One would expect here ‘lime of travertine,’ for what Vasari must mean is
lime prepared by burning this stone, which he recommends elsewhere, e.g.
‘Architettura,’ cap. iv, and ‘Scultura,’ cap. vi (calce di trevertino). The cement here
given is a lime cement mixed with water. A sort of putty mixed with boiled oil is
also employed, and is said to have been introduced by Girolamo Muziano of
Brescia, a contemporary of Vasari. Each mosaic worker seems to have his own
special recipe for this compound.
255. The process described by Vasari of building up the mosaic in situ, tessera
by tessera, according to the design pounced portion by portion on the soft cement,
is the most direct and by far the most artistic, and was employed for all the fine
mosaics of olden time. In modern days labour-saving appliances have been tried,
though it is satisfactory to know that they are all again discarded in the best work
of to-day, such as that of Sir W. B. Richmond in St. Paul’s. One of the methods
referred to, which can be carried out in the studio, is to take a reversed tracing of
the design, covered with gum, and place the cubes face downwards upon it
according to the colour scheme. When they are all in position, as far as can be
judged when working from the back, a coating of cement is laid over them and they
are thus fixed in their places. The whole sheet is then lifted up and cemented in its
proper place on the wall, the drawing to which the faces of the cubes are gummed
being afterwards removed by wetting. A better plan than this is called by the
Italians ‘Mosaico a rivoltatura.’ For this process the tesserae are laid, face upwards,
in a bed of pozzolana, slightly damp, which forms a temporary joint between the
adjacent cubes. Coarse canvas is pasted over the face of the work; it is lifted up,
and the pozzolana brushed out of the interstices. The whole is then applied to the
wall surface and pressed into the cement with which this has been coated. When
the cement has set the canvas is removed from the face.
256. The Duomo of Siena is a veritable museum of floor decorations in incised
outlines and in black and white, in the various processes described by Vasari.
There is a good notice of them in Labarte, Histoire des Arts Industriels. None of
the work is as early as the time of Duccio, but Beccafumi executed a large amount
of it. See the Life of that artist by Vasari.
It is worthy of notice that Dante had something of this kind in his thoughts,
when in the 12th Canto of the Purgatorio he describes the figure designs on the
ground of the first circle of Purgatory.

‘So saw I there ...

... with figures covered
Whate’er of pathway from the mount projects.

· · · · ·

O Niobe! with what afflicted eyes

Thee I beheld upon the pathway traced,
Between thy seven and seven children slain!
O Saul! how fallen upon thy proper sword
Didst thou appear there lifeless in Gilboa
That felt thereafter neither rain nor dew!
 · · · · ·

Whoe’er of pencil master was or stile,

That could portray the shades and traits which there
Would cause each subtle genius to admire?
Dead seemed the dead, the living seemed alive;
Better than I saw not who saw the truth,
All that I trod upon while bowed I went.’
Longfellow’s Translation.

257. See Note on ‘Tuscan Marble Quarries,’ ante, p. 119.

258. The Appartamento Borgia still contains a good display of these variegated
tiles; the original ones are however rather the worse for wear. In the Life of
Raphael, Vasari says they were supplied by the della Robbia of Florence. In the
Castle of S. Angelo there is a collection of interesting specimens of the tiles Vasari
goes on to mention. They are in cases in the Sala della Giustizia, and exhibit the
devices of Alexander VI, Julius II, Leo X, Paul III, and other Popes. The pavement
of the Laurentian Library at Florence is laid with tiles showing a very effective
design of yellow upon red. They are ascribed to Tribolo.
259. Was this the road from Seravezza seawards which Michelangelo had
begun? See Note on ‘Tuscan Marble Quarries,’ ante, p. 119. Specimens of these
Stazzema breccias are shown as C, D, on the Frontispiece.
260. Lat. Evonymus Europaeus. The only English example of the family is the
spindle tree.
261. The Lemonnier editors say that this work is lost. Of course Vasari is
speaking of the Old St. Peter’s, not the present structure.
262. Fra Damiano of Bergamo is mentioned by Vasari in his Life of Francesco
Salviati (Opere, ed. Milanesi, VII).
263. Inlays of different coloured woods, forming what is known as tarsia work,
and sometimes as marqueterie, compose an easily understood kind of decoration
that has been practised especially in the East from time immemorial. There is
however a special interest attaching to this work in the Italy of the fifteenth
century, in that it was connected with the studies in perspective that had so potent
an influence on the general artistic progress of the time. For some reason that is
not clearly apparent the designs for this work often took the form of buildings and
city views in perspective, and artists amused themselves in working out in this
form problems in that indispensable science. The history of the craft is so
instructive that it is worth a special Note, which the reader will find at the end of
this ‘Introduction,’ postea, p. 303.
264. ‘The onyx marbles of Algeria, Mexico, and California (which are of the
same nature as the Oriental alabasters) can be cut and ground sufficiently thin for
window purposes’ (Mr W. Brindley in Transactions, R.I.B.A., 1887, p. 53). See also
ante, p. 43.
265. The ‘occhi’ of Vasari correspond to the old-fashioned ‘bull’s-eyes’ which
are still to be seen surviving in cottage windows. The ‘bull’s eye’ pane was the
middle part of a sheet of so-called ‘crown’ glass where was attached the iron rod or
tube with which the mass of molten glass was extracted from the furnace, before,
by rotation of the rod, it was spread out into the form of a sheet. When the rod was
ultimately detached a knob remained, and this part of the sheet was used for
glazing as a cheap ‘waste product.’ In connection with the modern revival in
domestic architecture, for which Mr Norman Shaw deserves a good deal of the
credit, these rough panes have come again into fashion, and manufacturers make
them specially and supply them at the price of an artistic luxury! In Vasari’s time
they were evidently quite common, and we find numerous specimens represented
in the pictures of the fifteenth and sixteenth centuries. The bedroom of S. Ursula in
Carpaccio’s picture at Venice; the cell of S. Jerome in Dürer’s engraving; the room
in which van Eyck paints Arnolfini and his wife, those in which Jost Amman’s
‘Handworkers’ are busy, etc., etc., have casements glazed in this fashion, the knob,
called in English ‘bullion,’ in French ‘boudine,’ in German ‘Butzen,’ being distinctly
represented as in relief.
266. The ‘telajo di legno’ is a window frame of wood such as we are familiar
with in modern days, only in olden times these were often made detachable and
taken about from place to place when lords and ladies changed their domicile.
When Julius II wanted Bramante to fill some windows of the Vatican with coloured
glass, it was found that the French ambassador to the Papal court had brought a
painted window in such a frame from his own country, and the sight of this led to
the invitation to Rome of French artists in this material. See infra, Note 5.
267. See Note on ‘The Stained Glass Window’ at the close of this
‘Introduction,’ postea, p. 308.
268. Vasari wrote the life of this artist, who had been his own teacher in early
years at Arezzo (Opere, IV, 417). Gaye, Carteggio, II, 449, gives documentary
evidence that he was the son of a certain Pierre de Marcillat, and was born at S.
Michel in the diocese of Verdun in France. His name therefore has nothing to do
with Marseilles, which moreover is not in a glass-painting locality, whereas
Verdun, between France and Germany, is just in the region where the art was
developed and flourished. Guglielmo and another Frenchman named Claude came
to Rome about 1508 in the circumstances described in the foregoing Note, and
made some windows for the Sala Regia of the Vatican and other parts of the Palace.
These have all perished, but there still survive two windows from their hands in the
choir of S. Maria del Popolo, on which are the name and arms of Pope Julius II.
They are placed north and south behind and above the high altar, and have each
three lights. They contain scenes from the lives of Christ and the Madonna, in
which the figures are carefully drawn but the colour is patchy. Though the reds are
clear and strong, there is a good deal of grey and the architectural backgrounds are
rather muddy in hue. The artist was invited from Rome to Cortona and from
thence to Arezzo, which as Vasari notices in the beginning of his Life remained his
home to the end. He executed many windows there, in the cathedral and in S.
Francesco, some of which still remain; and also works in fresco. Vasari declares
that he owed to his teaching the first principles of art.
On the whole subject of the glass-painting craft see the Note on ‘The Stained
Glass Window,’ postea, p. 308, where the curious confusion of two different
processes, between which Vasari’s treatment oscillates, is elucidated.
269. The significance of Vasari’s demand for transparency in glass is explained
in the Note, postea, p. 308.
270. It is somewhat remarkable that the Venetians, who practised the art of
glass mosaic from about the ninth century, and in the thirteenth began their
famous glass works, never achieved anything in the technique of the stained glass
window. Venetian glass vessels, like the glorious lamps from the Cairo Mosques,
owe much of their beauty to the fact that the material is not clarified but possesses
a beautiful warm tone. It is indeed more difficult to get clear glass than tinted.
271. For the most part this description, with the exception of the part about
scaling-off glass in order to introduce a variety in colour, corresponds closely with
the technical directions which Theophilus gives so fully and clearly in his Schedula
Diversarum Artium of about 1100 A.D. It is pretty clear that Vasari is telling us here
what he learned from William of Marcillat who would have inherited the traditions
of the great French glass-painters of the thirteenth century.
272. The ‘scaglia’ is the thin scale that comes off heated iron when cooling
under the hammer, and is collected from the floors of smithies. Vasari thinks of it
as a ‘rust’ ‘ruggine,’ because rusty iron scales off in much the same way, the cause
in both cases probably being oxidization. Hence the expression ‘another rust.’
273. The pigments or pastes that are to be fused on to the coloured glass, to
modify its hue or to indicate details, are powdered and mixed with gum for
convenience in application. The gum is not to serve as permanent binding material
as the pastes are subsequently fused and burnt in on the glass.
274. It will be understood that the glass subjected to this treatment is not
coloured in the mass, or what is called ‘pot-metal,’ but has a film of colour ‘flashed’
or spread thinly on a clear sheet. This is done with certain colours, such as the
admired ruby red, because a piece coloured in the mass would be too opaque for
effect. Economy may also be a consideration, as the ruby stain is a product of gold.
275. The composition, which when fused stains the glass yellow, may before
fusion be of a red hue. As a rule the yellow stain on glass is produced by silver.
Vasari does not say what his composition is.
276. The red film is what Vasari understands by the ‘painting.’ This might fuse
and run with the heat required to fuse the yellow.
 277. That is, the space where the yellow leaf is to come may be cleared of the
red film after the yellow leaf has been painted on the back, as well as before that
process. The process Vasari describes of introducing small details of a particular
colour into a field of another hue is a good deal employed by modern workers in
glass, but it was not known to Theophilus, or much used in the palmy days of the
art, the twelfth and thirteenth centuries.
278. In Theophilus’s time these convenient leads grooved on both sides, which
are still in use, were not invented. He directs the worker to bind strips of lead
round each piece of glass and then solder together the leads when the pieces so
bound are brought into juxtaposition.
279. ‘Niello’ is from the mediaeval Latin ‘nigellum,’ ‘black,’ and refers to the
black composition with which engraved lines in metal plates were filled, according
to the process detailed by Vasari.
280. It is curious that the chapter ends without any discussion of the chasing
of gold and silver plate.
281. To some small extent the ancients do seem to have filled the engraved
lines in their bronze or silver plates with colouring matter, and the known
examples are described in Daremberg et Saglio, Dictionnaire des Antiquités, art.
‘Chrysographia,’ p. 1138. Pliny, Hist. Nat., XXXIII, 46, gives a recipe, as used by the
Egyptians, for a material for colouring silver that corresponds with the
composition used for niello work, though the use he indicates seems rather that of
an artificial patina than a filling for incisions. In any case the use of such a filling in
antiquity was quite uncommon, for the innumerable incised designs on the backs
of Greek and Etruscan mirrors and on caskets like the Ficeronian Cista show no
indication of the process, though of course in the lapse of time the incisions have
acquired a darker tinge than the smooth surfaces of the metal, and Vasari may
have seen them filled with accidental impurities.
282. A burin is shown in Fig. 2, D, ante, p. 48.
283. Vasari makes no mention here of sulphur, which in the recipes given by
Pliny, Theophilus, and Cellini, is a constant constituent of the black amalgam.
Silver and lead alone would not give the black required.
284. The ‘Pax,’ Italian ‘pace,’ was a little tablet of metal or some other material
used in churches to transmit the kiss of peace from the priest to the people. Certain
paxes once in the Baptistry of Florence have now found their way through the
Uffizi to the Museum in the Bargello, but experts are not agreed as to the
ascription of particular examples to Finiguerra. See Milanesi’s note on this artist at
the close of Vasari’s Life of Marc Antonio Raimondi (Opere, V, 443).
285. In Vasari’s first edition, of 1550, there is a notice of Finiguerra in the Life
of Antonio Pollaiuolo (p. 498) and he there celebrates only the skill of Maso as a
niellist, but in the edition of 1568 there is another notice of him in connection with
Marc Antonio (Opere, ed. Milanesi, V, 395), and here Vasari claims for him the
credit of being the first to make the advance from niello work to copper-plate
engraving. This second passage is a famous one, and describes how Finiguerra
moulded his silver plate, incised with a design, in clay, and then cast it in sulphur,
and subsequently filled the hollow lines in the sulphur cast (which reproduced the
incisions on the silver plate) with lamp-black, so that they showed up more clearly.
He then seems, according to Vasari, to have pressed damp paper against the
sulphur plaque so treated, and obtained a print by extracting the black from the
lines. Benvenuto Cellini however, a better authority than Vasari on Finiguerra,
praises him as the best niello worker of his time, but says nothing about this
further development of his craft, and on the contrary ascribes the invention of
copper-plate engraving to the Germans. Cellini tells us at the end of his
‘Introduzione,’ that in 1515, when fifteen years old, he began to learn the
goldsmith’s trade, and that then, though the art of niello work had greatly declined,
the older goldsmiths sang in his ears the praise of Maso Finiguerra, who had died
in 1464. Hence, Cellini says, he gave special attention to niello work, and he
describes the process, at rather greater length than Vasari, in the first chapter of
his Treatise on Gold-work (I Trattati, etc. di Benvenuto Cellini, ed. Milanesi,
Firenze, 1893).
The question of the origin of copper-plate engraving need not be here
discussed. Any of the incised silver or bronze plaques of the ancients might have
been printed from; and as a fact some incised bronze discs that are placed at the
bottoms of the towers in the great crown-light of the twelfth century in the Minster
at Aachen have actually been put through the printing press and the impressions
published, though no one at the time they were made can have thought of printing
from them. In the same way wooden stamps in relief were used by Egyptians and
Romans for impressing the damp clay of their bricks, though no one seems to have
thought of multiplying impressions on papyrus or parchment. So trial impressions
of niello plates, before the lines were filled in permanently, may often have been
made, and not by Finiguerra alone. The idea of multiplying such impressions on
their own account is now universally credited to the Germans, and this seems also
to have been the opinion of Cellini. See his ‘Introduzione.’
286. That is to say, the bottoms of cups or chalices. There are notices of
armorial insignia, enamelled at the bottom of cups of gold used by some of the
French kings, in Labarte, Histoire des Arts Industriels.
287. Giulio: a piece coined under Pope Julius II, of the same value as the
‘paolo,’ and equivalent to 56 centesimi, or about 5½d. of our money.
288. That is, the outlines of the different figures, ornaments, or other objects
executed in low relief on the metal. See the Note on ‘Vasari’s Description of Enamel
Work’ at the close of the ‘Introduction’ to Painting, postea, p. 311.
289. ‘The other kind’ probably refers to the incisions on the niello plates of
which he has been speaking. These are hollow, or in intaglio, whereas the work he
is here describing is in relief.
 290. ‘Si fermino col martello.’ The only practicable use of the hammer in
connection with enamels is to pound the lumps of vitreous paste to a more or less
fine powder, in which form they are placed over the metal. Theophilus, in chapter
53 of his third Book, ‘de Electro,’ ‘on Enamel,’ introduces the hammer in a similar
connection: ‘Accipiensque singulas probati vitri ... quod mox confringas cum
rotundo malleo donec subtile fiat;’ ‘take portions of the glass you have tested ... and
break up each lump with a round headed hammer till it be finely powdered.’ Cellini
also says the pastes are to be pounded in a mortar ‘con martello.’ Trattati, p. 30. It
is not easy however to see how any sense of ‘pounding’ can be extracted from the
verb ‘fermare’ which Vasari uses.
291. The difference in colour between gold and silver will naturally affect the
choice of the transparent vitreous pastes that are to cover them, and there are also
considerations of a chemical kind which prevent the use of certain pastes on
certain metal grounds. For example tin has the property of rendering transparent
enamels opaque, and transparent pastes cannot be used over metal grounds
wherein tin enters into the composition. Cellini, who gives the same caution as
Vasari, takes as an illustration transparent ruby coloured enamel, which he says
cannot be used over silver, for a reason which has about it a reminiscence of the
ancient alchemy, namely, that it is a product of gold and must be employed only
over its kindred metal! On the other hand he forbids for use with gold yellow,
white, and turquoise blue. We are indebted for some special information on this
highly technical subject to the kindness of Mr H. H. Cunynghame, C.B., who
writes: ‘There are two distinct reasons why different enamels are used on silver and
gold respectively. The first is an artistic reason. Transparent reds do not show well
over silver, the rays reflected from a silver surface not being well calculated to
show off the colours of the gold. In fact silver absorbs those rays on the
transmission of which the beauty of gold-red largely depends, whence then it
follows that transparent blues and greens should be used on silver, and reds,
browns, and the brighter yellows on gold. In addition to this, silver has its surface
disturbed by the silicic acid in the enamel. The consequence is that ordinary
enamels put on a silver surface are stained. To prevent this it is desirable to add
some ingredient that dissolves and renders colourless the stain. For this purpose
therefore special fluxes or clear enamels are made for silver. They usually contain
manganese and arsenic. The first of these has such a property of “clarifying”
enamels and glazes that it used to be called the potter’s “soap,” for it cleaned the
glazes on china. The other is also used for the same purpose.... As silver alloy is
more easy to melt than gold alloy, fluxes, i.e. clear enamels for silver, are much
more fusible than those for gold.’
292. This is a practice of modern enamellers. Cellini however is against it, as if
the enamels begin again to run there is a danger of losing the truth of the surface.
He recommends polishing by hand alone (Trattati, ed. Milanesi, 35).
293. This may have been the so-called Venetian enamel used in Vasari’s time.
This was a form of opaque painted enamel over copper, extremely decorative, but
coarse as compared with the translucent enamel over reliefs. We owe this
suggestion to Sir T. Gibson Carmichael.
294. The word ‘Tausia,’ and its connection with ‘Tarsia,’ the term used for
wood inlays, has given rise to some discussion. The explanation in Bucher’s
Geschichte der Technischen Künste, III, 14, is probably correct, and according to
this the Italian ‘Tausia’ comes from the Spanish ‘Tauscia’ or ‘Atauscia,’ which is
derived from an Arabic root meaning ‘to decorate.’ The art of inlaying one metal in
another is one of great antiquity in the East, and was no doubt brought to Spain by
the Moors, from which country, perhaps by way of Sicily, it spread to Italy. The
word ‘Tarsia,’ applied as we have already seen to inlays in wood, may have been
derived by corruption from ‘Tausia,’ though, as the form ‘Intarsia’ is also common,
a derivation (unlikely) has been suggested from the Latin ‘Interserere.’ The ‘in’ is
probably only the preposition, that has become incorporated with the word it
preceded.
295. ‘Cavasi il ferro in sotto squadra.’
296. If the sinkings be undercut the further process of roughening the sunk
surfaces is hardly necessary, but the roughening or puncturing may suffice to hold
the inlaid metal when there is no actual undercutting of the sides of the sinkings.
297. The ‘filiera,’ or iron plate pierced with holes of various sizes for drawing
wires through, was known to Theophilus. See chapter 8 of Book III of the Schedula,
‘De ferris per quae fila trahuntur.’
298. Vasari does not attempt to deal with the art of wood engraving in general
nor need this Note traverse the whole subject. In all these later chapters of the
‘Introduction’ to Painting he is dealing with forms of the decorative art in which
various materials are put together so as to produce something of the effect of a
picture. Hence all that he envisages in the department of wood engraving are what
are called chiaroscuri, or engravings meant to produce the effect of shaded
drawings by tints rather than by the lines which constitute engravings proper. It
has been noticed that some writers on engraving, (ante, p. 20) have denied to these
imitated light-and-shade drawings the character of true engravings.
As we have seen to be the case with copper-plate engraving (ante, p. 275)
priority is now claimed in these chiaroscuri for Germany over Italy, and Ugo da
Carpi, who was born about 1450, near Bologna, becomes rather the improver of a
German process than the inventor of a new one. On July 24, 1516, when resident in
Venice he petitions the Signoria of that city for privilege for his ‘new method of
printing in light and shade, a novel thing and not done before.’ Lippmann (The Art
of Wood Engraving in Italy in the Fifteenth Century, trans., London, 1888) thinks
that this claim may be true ‘in so far as he may have introduced further
developments in the practice of colour printing with several blocks, which still
survived in Venice, especially after the production of coloured wood-cuts by
Burgkmair and Cranach in Germany had given fresh stimulus to a more artistic
cultivation of that method’ (p. 69), and that ‘he gave the art an entirely new
development based upon the principles which guided the profession of painting’
(p. 136). This last phrase explains the interest that Vasari here manifests in his
work. In the older wood engraving only lines had been left on the block to take the
ink, the rest of the surface being cut away, and whatever was to be shown in the
print was displayed in the lines alone. In the new method broad surfaces of the
wood were left, on which was spread a film of ink or pigment, and these printed a
corresponding tint upon the paper which took off the film thus laid. The pigment
might be of any colour desired, or might only represent a lighter tint of the ink that
had been used all along for the lines. Hence either an effect of colour or one merely
of gradations of light and shade could equally well be produced by the process
Vasari describes. The work he contemplates is of the latter kind, and his
explanation of the process by which it was produced is fairly clear. Plate XIV, from
a print by Ugo da Carpi in the British Museum, gives a specimen of the result.
Critics of Ugo da Carpi’s work, which is sufficiently abundant, notice that he
begins by merely adding tints of shading to outlines, which as in the earlier
productions of the Germans, like those of Cranach or Dienecker, remained
substantially responsible for the effect; but that he gives more and more
importance to the tints, the pictorial element in the design, till the outlines end by
merely reinforcing the chiaroscuro, like the touches ‘a tempera’ that give effect and
decision to painting in fresco (Kristeller, Kupferstich und Holzschnitt in vier
Jahrhunderten, Berlin, 1905, p. 300).
299. That is, he made three blocks A, B, C, each the full size of the design, but
each containing only a part of the work. A has engraved on it all the lines of the
design, and a print from it would be an old-fashioned engraving proper. Such a
print with the ink on it still wet is pressed down on a clean block of wood, on which
it leaves indications of all these lines. The broad tints of shading, in which
gradations may be introduced, are then laid on the block by hand, the outlines
being a guide, and so is constituted block B, an impression from which printed on a
sheet already printed from block A, and made to register accurately with this,
would add shading to the outlines. C would add by the same process a third tint,
quite flat, for the background, and this might of course be of another colour. The
high lights would be cut away in this block, C, and these parts come out white in
the print, as is seen on Plate XIV. The uniform grey shade on the Plate is the
background tint. In the actual process of printing this block, C, is first put into the
press and produces an impression showing the tinted background but white spaces
where the high lights are to come. B, with the shadows tinted but all the rest of the
wood cut away, is printed over the impression from C, and lastly A comes to give
the decided lines and sharpen up the whole effect.
300. The ‘oil colour’ is the pigment which is transferred from the block to the
paper. The ‘water colour’ and the ‘white lead mixed with gum’ mentioned above are
only put on by the artist to guide the wood-cutter in his work of cutting the block.
301. The text, in both the original editions, runs as follows: ‘E la terza che è la
prima a formarsi, è quella dove il profilato del tutto è incavato per tutto, salvo che
dove e’ non ha i profili tocchi dal nero della penna,’ and the negative is puzzling,
for obviously the wood must be cut away everywhere but in those places where the
outlines do come.
302. But Theophilus says practically nothing about design, and yet the
mediaeval epoch was for the decorative arts one of the most glorious the world has
ever seen. See on this subject the last part of the Introductory Essay, ante, p. 20 f.
 TRANSCRIBER’S NOTES
1. Typos fixed; non-standard spelling and dialect retained.
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