Pyrosequencing: Methods and Protocols

429 Pages·2015·12.568 MB·English

by  Ulrich Lehmann, Jörg Tost

Methods in
Molecular Biology 1315
Ulrich Lehmann
Jörg Tost Editors
Pyrosequencing
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Pyrosequencing

Methods and Protocols
Second Edition
Edited by
Ulrich Lehmann
Medizinische Hochschule Hannover, Institute of Pathology, Hannover, Germany
Jörg Tost
Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique,
Evry, France
Editors
Ulrich L ehmann Jörg T ost
Medizinische Hochschule Hannover Laboratory for Epigenetics and Environment
Institute of Pathology Centre National de Génotypage
Hannover, Germany CEA-Institut de Génomique
Evry, France
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2714-2 ISBN 978-1-4939-2715-9 (eBook)
DOI 10.1007/978-1-4939-2715-9
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Prefa ce
The Pyrosequencing technology has been around for nearly 20 years and has become a
widely used technology for a broad variety of applications to analyze genetic and epigenetic
variation as well as to identify bacteria, viruses, and microorganisms. Pyrosequencing is a
versatile, fl exible, accurate, and extremely quantitative sequencing-by-synthesis technology,
which is relatively easy to handle and has a short time to results. The sequence-based read-
out, and the rapid and standardized work-fl ow of Pyrosequencing as well as the improved
limit of detection and analytical sensitivity compared to traditional sequencing technolo-
gies, make this technology a method of choice for diagnostic applications. Furthermore, it
has established itself as the “gold standard” platform for quantitative locus-specifi c DNA
methylation analysis.
Eight years have passed since the fi rst edition of this volume edited by Sharon Marsh in
2007 and genomics and medical diagnostics have undergone a major revolution in the time
from the last edition. While Pyrosequencing has been used as the underlying principle for
the fi rst commercially available next-generation sequencing system (454 Life Sciences, now
part of Roche Molecular Diagnostics), the technology has not been able to evolve as rapid
as other sequencing platforms. However, its use in locus-specifi c analyses is still expanding
and novel applications are continuously devised. Despite the rapid advancements in the
fi eld of comprehensive high-throughput genome-wide “omics” technologies, declining
costs, and the development of more user-friendly systems, we are convinced that fast, cost-
effective, focused, robust, and widely distributed technologies with a moderate through-
put, like Pyrosequencing, will have their place for the foreseeable future, not only for the
independent validation of fi ndings from genome-wide screening approaches but also for
the exact quantitative analysis of single (or only a few) loci and within the molecular diag-
nostic laboratory.
The second edition of the “Pyrosequencing Protocols” contains 27 exciting chapters
written by experts in the fi eld that use this platform for their different analyses. With the
exception of the chapter describing the history of Pyrosequencing and its development by
Pal Nyren, one of the inventors of the technology who updated his historical overview, all
other chapters have been newly written for this edition and we hope to cover a broad range
of application. Chapters 1 – 4 provide a general overview on the biochemistry, the available
instruments, tools for the interpretation of the sequencing output (Pyrograms), as well as
general considerations on the strength and limitations of the technology that are of interest
to all applications of the technology. Chapters 5 – 12 focus on the quantitative analysis of
genetic variation, including detection of mutations of clinical relevance in different biologi-
cal specimens (Chapters 5 – 8 ) , copy number variation (Chapter 9 ) , and large-scale chromo-
somal alterations (Chapter 10 ). Chapter 1 1 describes the specifi c genotyping of HLA alleles
using Pyrosequencing, while Chapter 12 investigates the ratio of expressed alleles at the
RNA level. Chapters 13 – 22 focus on the analysis of DNA methylation including gene-
specifi c (Chapters 13 and 14 ) and global DNA methylation assays (Chapters 1 5 and 16 ) .
Specialized applications for DNA methylation analysis described in this volume include
v
vi Preface
single molecule analysis (Chapter 17 ) , loss of imprinting (Chapter 1 8 ) , single blastocyst
analysis (Chapter 19 ) , allele-specifi c DNA methylation patterns (Chapters 2 0 and 21 ) , as
well as the analysis of DNA methylation patterns associated with specifi c histone modifi ca-
tions (Chapter 22 ). Tools and protocols for the detection of viruses and bacteria are
described in Chapters 2 3 – 25 , while the exciting possibilities of genetic and epigenetic anal-
yses for forensics using Pyrosequencing are described in Chapters 26 and 2 7 .
This volume of the “Methods in Molecular Biology” series is addressed to advanced
doctorial students and postdoctoral investigators and research scientists that are implicated
in the different aspects of genetics and cellular and molecular biology as well as to clinicians
involved in diagnostics or choice of treatment of diseases that have an analytical component.
The presentation in this volume is equally suited for laboratories that already have a great
deal of expertise in Pyrosequencing, but might want to extend the use of this technology to
other applications, and for genetics/genomics/biology groups that want to initiate research
projects making use of the advantages of the Pyrosequencing technology. Notes and tips
from the experts for the different methods will enable a rapid implementation of the differ-
ent protocols in the laboratory and avoid time-consuming and cost-intensive mistakes.
We are indebted to all the authors for their hard work and outstanding contributions
to this second edition of “Pyrosequencing Protocols.” It was a pleasure to work with them
on this project. The series editor John Walker provided essential initial guidance, and the
staff from Springer was helpful in fi nalizing the publication process. We would also like to
thank Britta Hasemeier for her great help in harmonizing all artwork and reference lists
produced by more than 20 different groups.
We sincerely hope that the protocols described in detail in this volume will enable to
develop new applications of the Pyrosequencing technology increasing further the great
versatility of the highly accurate and quantitative technology and will help to enhance our
understanding of the molecular processes underlying biological processes and disease for
the benefi t of patients.
Hannover, Germany Ulrich L ehmann
Evry, France J örg T ost
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
PART I INTRODUCTION
1 The History of Pyrosequencing®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Pål Nyrén
2 PyroMark® Instruments, Chemistry, and Software
for Pyrosequencing® Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Martin Kreutz , Gerald Schock , Julia K aiser ,
Norbert H ochstein , and Ralf P eist
3 Software-Based Pyrogram® Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Guoli Chen , Matthew T. O lson , and James R. E shleman
4 Q uantitative Validation and Quality Control of Pyrosequencing® Assays . . . . . 3 9
Ulrich Lehmann
PART II ANALYSIS OF GENETIC VARIATION
5 Extended K RAS and NRAS Mutation Profiling by Pyrosequencing® . . . . . . . 4 9
Andreas Jung
6 U niversal BRAF State Detection by the Pyrosequencing®-Based
U-BRAFV600 Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Alexander Skorokhod
7 P yrosequencing®-Based Identification of Low-Frequency
Mutations Enriched Through Enhanced- ice- COLD-PCR . . . . . . . . . . . . . . . . 8 3
Alexandre How-Kit and Jörg T ost
8 Analysis of Mutational Hotspots in Routinely Processed
Bone Marrow Trephines by Pyrosequencing® . . . . . . . . . . . . . . . . . . . . . . . . . 1 03
Stephan Bartels and Ulrich L ehmann
9 A nalysis of Copy Number Variation by Pyrosequencing®
Using Paralogous Sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Marianne K ristiansen K ringen
10 P renatal Diagnosis of Chromosomal Aneuploidies by Quantitative
Pyrosequencing®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Hui Y e , H aiping W u , Y unlong Liu , Bingjie Zou ,
Tomoharu Kajiyama , Hideki K ambara , and Guohua Zhou
11 H LA-B and HLA-C Supratyping by Pyrosequencing®. . . . . . . . . . . . . . . . . . . 133
Irene Vanni , Elisabetta U golotti , Patrizia L arghero ,
and Roberto Biassoni
vii
viii Contents
12 Allele Quantification Pyrosequencing® at Designated SNP Sites
to Detect Allelic Expression Imbalance and Loss-of-Heterozygosity . . . . . . . . 153
Chau-To Kwok and Megan P . Hitchins
PART III ANALYSIS OF DNA METHYLATION PATTERNS
13 Quantitative DNA Methylation Analysis by Pyrosequencing® . . . . . . . . . . . . . 1 75
Jessica R oessler and U lrich L ehmann
14 Quantitative Methylation Analysis of the P CDHB Gene Cluster . . . . . . . . . . . 1 89
Barbara Banelli and Massimo R omani
15 Assessment of Changes in Global DNA Methylation Levels
by Pyrosequencing® of Repetitive Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Ali M. T abish , Andrea A . Baccarelli , L ode G odderis ,
Timothy M. B arrow , P eter H oet , and Hyang-Min Byun
16 G lobal Analysis of DNA 5-Methylcytosine Using the Luminometric
Methylation Assay, LUMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Karin Luttropp , Louise K. S jöholm , and Tomas J. Ekström
17 Limiting Dilution Bisulfite Pyrosequencing®: A Method
for Methylation Analysis of Individual DNA Molecules
in a Single or a Few Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 21
Nady El H ajj , Juliane Kuhtz , and Thomas H aaf
18 D etection of Loss of Imprinting by Pyrosequencing®. . . . . . . . . . . . . . . . . . . . 241
Silvia Tabano , E leonora Bonaparte , and M onica Miozzo
19 A nalysis of DNA Methylation Patterns in Single Blastocysts
by Pyrosequencing® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 59
John H untriss , Kathryn W oodfine , Joanna E . Huddleston ,
Adele M urrell , and Helen M . Picton
20 A llele-Specific DNA Methylation Detection by Pyrosequencing®. . . . . . . . . . . 271
Lasse S ommer K ristensen , Jens Vilstrup Johansen ,
and Kirsten Grønbæk
21 S NP-based Quantification of Allele-specific DNA Methylation
Patterns by Pyrosequencing®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Florence B usato and Jörg T ost
22 DNA Methylation Analysis of ChIP Products at Single Nucleotide
Resolution by Pyrosequencing®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Céline Moison , F anny A ssemat , Antoine Daunay , P aola B. Arimondo ,
and J örg Tost
PART IV BACTERIAL TYPING AND IDENTIFICATION
23 Multiplex Pyrosequencing®: Simultaneous Genotyping Based
on SNPs from Distant Genomic Regions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Piotr Wojciech D abrowski , Kati Bourquain , and Andreas Nitsche
24 D etection of Drug-Resistant M ycobacterium tuberculosis . . . . . . . . . . . . . . . . . 3 49
Anna E ngström and Pontus J uréen
25 A pplication of Pyrosequencing® in Food Biodefense . . . . . . . . . . . . . . . . . . . . 3 63
Kingsley Kwaku Amoako
Contents ix
PART V FORENSICS
26 Forensic Analysis of Mitochondrial and Autosomal Markers
Using Pyrosequencing®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Magdalena M . B uś , Hanna E dlund , and M arie Allen
27 T issue-Specific DNA Methylation Patterns in Forensic
Samples Detected by Pyrosequencing® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 97
Joana Antunes , K uppareddi Balamurugan , G eorge Duncan ,
and Bruce McCord
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 11