Phylogenomic Data Acquisition: Principles and Practice, 1st Edition (Hardback) book cover

Phylogenomic Data Acquisition

Principles and Practice, 1st Edition

By W. Bryan Jennings

CRC Press

232 pages | 30 Color Illus. | 56 B/W Illus.

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Hardback: 9781482235340
pub: 2016-11-23
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Phylogenomics is a rapidly growing field of study concerned with using genome-wide data—usually in the form of DNA sequence loci—to infer the evolution of genes, genomes, and the Tree of Life. Accordingly, this discipline connects many areas in biology including molecular and genomic evolution, systems biology, molecular systematics, phylogeography, conservation genetics, DNA barcoding, and others. With the advent of Next Generation Sequencing in addition to advances in computer hardware and software over the past decade, researchers can now generate unparalleled phylogenomic datasets that are helping to illuminate many areas in the life sciences. This book is an introduction to the principles and practices of gathering these data. Phylogenomic Data Acquisition: Principles and Practice is intended for a broad cross-section of biologists and anyone else interested in learning how to obtain phylogenomic data using the latest methods.


"There is definitely a need for this book written in the language of the phylogeneticist. The general organization of the book is good. This book addresses a broad range of topics in a way that makes them comprehensible to people who are not experts in bioinformatics and will be extremely influential. Jennings has been in on the ground floor of the multilocus coalescent revolution in phylogenetics."

Jim McGuire, University of California Berkeley

"Genome sequencing technologies and data collection techniques are changing rapidly, and this book is a great resource for getting up to speed in this exciting field. Jennings outlines the essential data collection strategies that all students entering the field of molecular systematics need to know. "

Adam Leaché, University of Washington, Seattle

"This new book by evolutionary biologist Bryan Jennings brings together a wealth of useful information allowing researchers to move from start to finish in the rapidly changing field of phylogenomics. It details step-by-step protocols and provides masterful summaries of best practices for the modern phylogeneticist. Graduate students, postdoctoral fellows and principal investigators will find this book extremely useful. Jennings cuts through the jungle of sometimes conflicting information and provides cogent reviews of important topics and guidelines for avoiding common pitfalls."

Scott Edwards, Harvard University

Table of Contents


What Is Phylogenomics?

The Early View of Phylogenomics

An Expanded View of Phylogenomics

Anatomy of Gene Trees

Gene Trees vs. Species Trees

Phylogenomics and the Tree of Life

Sequencing Workflows to Generate Phylogenomic Data

Sanger Sequencing Workflow

Next Generation Sequencing Workflow

Is Sanger Sequencing Still Relevant in Phylogenomics?

The Phylogenomics Laboratory


Genomic Background

Genome Types and Sizes

Composition of Eukaryotic Organellar Genomes

Composition of Eukaryotic Nuclear Genomes

Gene Numbers and Densities among Nuclear Genomes

Intergenic DNA

DNA Sequence Evolution

Patterns and Processes of Base Substitutions

Transition Bias

Transition Bias and DNA Replication Errors

Saturation of DNA Sites

Among-Site Substitution Rate Variation

Tandemly Repeated DNA Sequences

Transposable Elements

Processed Pseudogenes

Mitochondrial Pseudogenes ("Numts")

Numt Abundance in Eukaryotic Genomes

Mechanisms of Primary Numt Integration

Differences between Numts and Mitochondrial DNA


Six Assumptions about DNA Sequence Loci in Phylogenomic Studies

Assumption 1: Loci Are Single-Copy in the Genome

Assumption 2: Loci Are Selectively Neutral

Does "Junk DNA" Exist?

The Neutrality Assumption and the Indirect Effects of Natural Selection

Assumption 3: Sampled Loci Have Independent Gene Trees

How Many Independent Loci Exist in Eukaryotic Genomes?

Criteria for Delimiting Loci with Independent Gene Trees

Assumption 4: No Historical Recombination within Loci

Intra-Locus Recombination and Gene Trees

What Is the Optimal Locus Length?

Assumption 5: Loci Evolved Like a Molecular Clock

Assumption 6: Loci Are Free of Ascertainment Bias

DNA Sequence Loci: Terminology and Types

On Genes, Alleles, and Related Terms

Commonly Used DNA Sequence Loci in Phylogenomic Studies

Mitochondrial DNA Loci

Nuclear DNA Loci


DNA Extraction Methodology

Summary of the DNA Extraction Process

A Note about DNA Storage Buffers

Extracting DNA from Plants, Fungi, and Invertebrates

Extracting DNA from Formalin-fixed Museum Specimens

Evaluating the Results of DNA Extractions

Agarose Gel Electrophoresis


UV Spectrophotometric Evaluation of DNA Samples

UV Spectrophotometry to Determine Concentrations of Nucleic Acid Samples

UV Spectrophotometry to Determine the Purity of DNA Samples

Fluorometric Quantitation of DNA Samples

The High Throughput Workflow

High Throughput DNA Extractions

Extracting DNA from 96 Tissue Samples

High Throughput Agarose Gel Electrophoresis

High Throughput UV Spectrophotometry

Preparation of Diluted DNA Templates for High Throughput PCR


Historical Overview

DNA Polymerization in Living Cells vs. PCR

Brief Review of DNA Polymerization in Living Cells

How the Polymerase Chain Reaction Works

PCR Procedures

Preparation of PCR Reagents and Reaction Setup

PCR Reagents

Importance of Making Reagent Aliquots

Setting up PCR Reactions


Checking PCR Results Using Agarose Gel Electrophoresis

PCR Troubleshooting

Reducing PCR Contamination Risk

High Throughput PCR

Setting up PCR Reactions in a 96-sample Microplate Format

Other PCR Methods

Hot Start PCR

Long PCR



Principles of Sanger Sequencing

The Sanger Sequencing Concept

Modern Sanger Sequencing

Cycle Sequencing Reaction

Gel Electrophoresis of Extension Products

Sequence Data Quality

Sanger Sequencing Procedures

Purification of PCR Products

Exo-SAP Treatment of PCR Products

Spin Column and Vacuum Manifold Kits for PCR Product Purification

20% PEG 8000 Precipitation of PCR Products

Solid-Phase Reversible Immobilization (SPRI) Beads

Gel Purification of PCR Products

Which PCR Product Purification Method Is Best?

Setting up Cycle Sequencing Reactions

Purification of Extension Products

Sequencing in a Capillary Sequencer: Do-it-Yourself or Outsource?

High Throughput Sanger Sequencing

Sequencing 96 Samples on Microplates

Adding Sequencing Primer "Tails" to PCR Primers

How an M13-tailed Primer Functions in PCR

Cycle Sequencing and M13 Primer Tails

On the Importance of Matching Sequencing Primers

Benefits of Using M13-Tailed Primers

Haplotype Determination from Sanger Sequence Data

PCR Amplification and Sanger Sequencing of Diploid or Polyploid Loci

Multiple Heterozygous SNP Sites and Haplotype Sequences

Methods for Obtaining Nuclear Haplotype Sequences from Sanger Sequence Data

Physical Isolation of PCR Haplotypes Prior to Sequencing

Statistical Inference of Haplotypes from Sanger Sequence Data


How Illumina Sequencing Works

Construction of Indexed Sequencing Libraries

Generation of Clusters on the Flow Cell

Sequencing of Clusters

Methods for Obtaining Multiplexed Hybrid Selection Libraries

Library Preparation Approaches

Traditional Illumina Library Approach

Meyer and Kircher Library Approach

Rohland and Reich Library Approach

Nextera Library Approach

In-Solution Hybrid Selection

Indexing, Pooling, and Hybrid Selection Efficiency Revisited

Cost-effective Methods for Obtaining Multiplexed Targeted-Loci Libraries

Sequence Capture Using PCR-generated Probes (SCPP)

Parallel Tagged Amplicon Sequencing (PTS)


Primer Design Theory

Rules of Primer Design

Final Comments about Primer Design Rules

Testing New Primers in the Lab

Primer and Probe Design Approaches

Single Template Approaches for Developing PCR-based Loci

Single Template Methods Using Genomic Cloning Methods

Single Template Methods Using Available Genomics Resources

Single Template Methods Using NGS Partial Genome Data

Single Template Methods Using Whole Genome Sequences

Multiple Homologous Template Approaches for Designing PCR-based and Anchor Loci

Designing Universal Primers by Comparative Sequence Analysis

Multiple Homologous Template Approaches Using Whole Genome Sequences

Designing Anchor Loci Probes Using Whole Genome Sequences


The Impending Flood of Genomes

In Silico Acquisition of Phylogenomic Datasets

About the Author

W. Bryan Jennings is professor and coordinator for the Molecular Laboratory of Biodiversity Research at Universidade Federal do Rio de Janeiro.

Subject Categories

BISAC Subject Codes/Headings:
SCIENCE / Life Sciences / Evolution
SCIENCE / Life Sciences / Genetics & Genomics
SCIENCE / Research & Methodology
SCIENCE / Life Sciences / General