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Learn more about
the most published
next generation
sequencing
platform.
Bibliography
April 18, 2008
Genome Sequencer FLX System
More Flexibility, More Applications, More Publications
www.roche-applied-science.com
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Genome Sequencer FLX System
More flexibility, more applications
The Genome Sequencer FLX System is the newest addition to Roche’s next-generation
sequencing platform, building upon proven technology and peer-reviewed publications.
■
Generate over 100 million bases per 7.5-hour instrument run.
■
Achieve longer reads, averaging 250 to 300 bases.
■
Attain higher throughput with over 400,000 reads per run.
■
Generate single-read accuracy that is greater than 99.5% over 200-base reads.
■
Benefit from consensus accuracy that is greater than 99.99%.
The Genome Sequencer FLX System features complete software packages, including
easy-to-use graphical user interfaces (GUI) for mapping, assembly, and amplicon
variation detection.
■
Use the project management software tool to group multiple instrument runs for analysis,
and add instrument runs over time for additional analysis power.
■
Obtain higher-confidence results using next-generation algorithms — from base calling to
assembly and mapping, along with variation analysis.
Perform breakthrough science
with more applications using
the flexibility of the
Genome Sequencer FLX System.
■
De Novo Sequencing Supported by
Shotgun and Paired-End Applications
■
Comparative Genomics using
Whole Genome Sequencing
■
Amplicon Resequencing
■
Transcriptome Analysis
■
Gene Regulation Studies
■
ChIP-Sequencing
Genome Sequencer
FLX System
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Genome Sequencer FLX System Workflow
One fragment = One bead = One read
The Genome Sequencer FLX System provides a complete solution —
from sample preparation through digital data analysis.
1) Sample Input: The Genome Sequencer FLX System supports the
|——————————— x 400,000 + ———————————|
sequencing of samples from a variety of starting materials, including
genomic DNA, PCR products, BACs, and cDNA.
2) Sample Fragmentation: Samples such as genomic DNA and BACs are
fractionated into small 300 to 800 base-pair fragments. For some samples,
such as small non-coding RNA, fragmentation is not required. Short PCR
products can be amplified using Genome Sequencer fusion primers to go
directly to Step 4, shown below.
3) Adaptor Ligation: Using a series of standard molecular biology
techniques, short adaptors (A and B) – specific for both the 3' and 5'
ends – are added to each fragment. The adaptors will also be used for
purification, amplification, and sequencing steps. Single-stranded
fragments, shown here, are used in subsequent steps in the workflow.
4) One Fragment = One Bead: The first step in emulsion PCR is shown.
The adaptors enable hundreds of thousands of single-stranded fragments
to bind to their own unique beads. The beads are then encapsulated into
individual micelles formed by a water-in-oil emulsion, creating a
micro-reactor containing one bead with one unique fragment. Each
unique fragment is amplified without the introduction of competing or
contaminating sequences. The entire fragment collection is amplified in
parallel.
5) Clonally Amplified Bead: The PCR process amplifies each fragment to
a copy number of several million per bead. Subsequently, the emulsion
PCR is broken while the fragments remain bound to their specific beads.
After enrichment, the clonally amplified bead is ready to load onto the
PicoTiterPlate device for sequencing.
6) Flowgram: A digital output of the real-time sequencing-by-synthesis.
Each flowgram represents the sequence read from one bead which has one
unique amplified fragment attached to it. The standard Genome Sequencer
FLX System run produces over 400,000 flowgrams or reads per 7.5-hour
instrument run. Flowgrams can be exported as .SFF files for submission to
NCBI.
Figure 1: Genome Sequencer FLX System Workflow Overview.
www.genome-sequencing.com
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Sequencing Chemistry
The Technology
454 Sequencing, based on sequencing-by-synthesis (Figure 1), is the power
behind the performance of the Genome Sequencer FLX System.
Sequencing-by-Synthesis: Using an enzymati-
Nucleotides are flowed sequentially in a fixed order across the
PicoTiterPlate device during a sequencing run. During the nucleotide
flow, hundreds of thousands of beads each carrying millions of copies of a
unique single-stranded DNA molecule are sequenced in parallel. If a
nucleotide complementary to the template strand is flowed into a well, the
polymerase extends the existing DNA strand by adding nucleotide(s).
Addition of one (or more) nucleotide(s) results in a reaction that generates
a light signal that is recorded by the CCD camera in the instrument.
cally coupled reaction, light is generated when
individual nucleotides are incorporated. Hundreds
of thousands of individual DNA fragments are
The signal strength is proportional to the number of nucleotides
incorporated in a single nucleotide flow.
sequenced in parallel.
Multiplex Identifier Kits (MID)
Increasing sample throughput per instrument run
Uniquely tag up to twelve samples with new ligation Multiplex Identifiers (MIDs), reducing the cost per
sample, increasing multiplexing capabilities, and simplifying the handling of multiple samples. Twelve
MID adaptors are available, each containing a unique 10-nucleotide sequence. During library preparation,
sample-specific MID adaptor molecules are blunt-end ligated onto each DNA fragment in a sample. The
MID-tagged samples can be pooled for simultaneous amplification and sequencing. Used in conjunction with
the available gasket, up to 192 samples can be sequenced per run. Each MID is recognized by the GS FLX
analysis software, allowing for automated grouping and analysis of MID-containing reads.
3|
■
Simplify sample preparation by eliminating the need for individual amplification reactions.
■
Expand experimental design options by multiplexing samples.
■
Obtain more reads per sample by reducing the need for gaskets, making more PicoTiterPlate wells available
for sequencing.
■
Reduce the risk of contamination by tagging each DNA molecule with a sample-specific MID.
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Sequencing Kits for the Genome Sequencer FLX System
Sequencing PicoTiterPlate
Kit
Device
GS LR70
70 x 75
GS SR70
70 x 75
GS LR25
25 x 75
Number of Regions
per Gasket
Read
Length
(bases)
2 large regions
4 medium regions
8 medium regions
16 small regions
2 large regions
4 medium regions
8 medium regions
16 small regions
240
240
240
240
100
100
100
100
1 medium region
4 small regions
240
240
Run
Time
Number of
Reads per
Region
7.5 hrs
4 hrs
7.5 hrs
Number of
Bases per
Region
Total Number
or Reads
per Gasket
Total Number
or Bases
(all regions)
210,000
70,000
30,000
12,000
210,000
70,000
30,000
12,000
50.4 Mb
16.8 Mb
7.2 Mb
2.88 Mb
21 Mb
7 Mb
3 Mb
1.2 Mb
420,000
280,000
240,000
192,000
420,000
280,000
240,000
192,000
100.8 Mb
67.2 Mb
57.6 Mb
46.08 Mb
42 Mb
28 Mb
24 Mb
19.2 Mb
70,000
12,000
16.8 Mb
3 Mb
70,000
48,000
16.8 Mb
24 Mb
Table 1. Number or reads and bases per PicoTiterPlate device configuration. Data was generated using genomic DNA from E. coli.
Results may vary depending upon type of sample and organism, average read length observed for a specific genome, or application.
(Mb = million bases)
Recommended Applications
Applications for GS LR70
Applications for GS SR70
Applications for GS LR25
■
Whole genome sequencing
■
Small RNA
■
Titrations
■
Metagenomics
■
Expression profiling
■
Amplicons for resequencing
■
Long amplicon resequencing
■
Short amplicons (up to 100
pairs)
■
BACs
■
Shotgun sequencing of small
genomes (virses, small bacteria,
etc.)
Configure the PicoTiterPlate Device using various gaskets
More flexibility with different sample loading options
For projects that require a higher number of samples with fewer sequencing reads per sample, a series of gaskets
are provided for both the small and large PicoTiterPlate devices. These gaskets provide physical barriers to divide
the area on the PicoTiterPlate device into smaller segments. When the gaskets are combined with the MID’s, a
larger number of samples can be sequenced in one instrument run.
Figure Legend: PicoTiterPlate and gaskets for GS LR25
Figure Legend: PicoTiterPlate and gaskets for GS LR70 and
reagents supporting 1 and 4 samples
GS SR70 reagents supporting 1/2, 4, 8 , and 16 sample configurations.
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Genome Sequencer FLX System
Workflow and Bioinformatics
T
Image
Capture
▼
Data
Processing
A
C
G
T
Data Transfer
to External PC
▼
Data
Processing
Image Files:
12-15 gigabytes
per run
Genome Sequencer FLX Instrument
Genome Sequencer FLX Instrument
■
Linux server unit and software for running the instrument (Uninterruptible Power Supply [UPS] unit can run the
instrument for up to 20 minutes in the event of power failure)
■
Software package on CD, which must be installed on an external PC.1 Package includes:
— Data processing software: image and signal processing, base calling, and quality scoring
— Application software
External PC and Data Storage Requirements (not supplied)
Computer requirements
64-bit dual processor (dual x86 CPU or better), 8 GB RAM,
500 GB hard drive or 32-bit dual processor, 4 GB RAM
Operating system
Red Hat Enterprise Linux 4 Workstation OS
(32-bit and/or 64-bit version) WS Standard
Java 1.5.0 or higher
5|
1
2
3
4
5
Ethernet
1 Gigabit Ethernet
Raw data output (image files)
12-15 gigabytes of data per run using a 70x75 PicoTiterPlate device
Processed data output
(Data Analysis Folder)
Up to 3 gigabytes per run using a 70x75 PicoTiterPlate device
Data storage requirement 2
(for data backup)
Backup of raw data such as image files is recommended
(e.g., 400 gigabytes per tape drive). Raw data can be reprocessed
using software updates.
There is no limitation to the number of installations in external PCs, and there are no registration codes.
Data storage requirement for the analyzed data depends on the number and type of analyses and project size.
GS Reference Mapper - amount of time required is comparable to that needed for GS De Novo Assembler for the same amount of data.
The time needed for data analysis using the different software tools strongly depends on project size and amount of data.
Approximately 30,000 reads are mapped from 11 amplicons in 1.5 minutes.
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Applications
De Novo
Assembly
Mapping
Amplicon
Analysis
Flowgram (read sequence)
Application Software
■ GS Reference Mapper
■ GS De Novo Assembler
■ GS Amplicon Variant Analyzer
Application Software
Data output
GS De Novo Assembler
■
De novo assembly of whole genomes (up to 120 megabases)
• fna.file (sequence of contigs, FASTA format)
■
Assembly of approximately 400,000 reads in 10 - 15 minutes
■
Addition of 20 - 100 bp paired-end reads to order and
orient the contigs into scaffolds
• qual.file (corresponding Phred equivalent
quality score)
• ace.file (alignment of the reads to contig
sequences)
GS Reference Mapper
■
Resequencing of microbial and eukaryotic genomes
(up to 3 gigabases) 3,4
■
A given reference sequence in FASTA.file format can be
compared with the sequence results
■
High-confidence sequence differences are listed
• fna.file (sequence of contigs, FASTA format)
• qual.file (corresponding Phred equivalent
quality score)
• ace.file (consensus alignment of the reads
against a given reference sequence)
GS Amplicon Variant Analyzer
■
Sequencing of PCR products with ultra-deep coverage
depth (e.g., identification of somatic mutations) 4,5
• ace.file (alignment of the reads against
a reference sequence)
■
Identification and quantification of sequence variations
■
Calculation of frequency for known variants and discovery
of new variants
• png.file (graphical file format, tab delimited
text file)
Data output compatibility
■
Data format is compatible with different publicly available tools (e.g., consed and data archive such as NCBI).
■
For applications (e.g., metagenomics) not supported by our software modules, publicly available tools can be used
by bioinformatics specialists.
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3K Long-Tag Paired End Sequencing with the Genome
Sequencer FLX System
The 3K Long-Tag Paired End library DNA fragments contain an approximately 250-bp fragment with a 44-mer
adaptor sequence in the middle, flanked by 100-mer sequences, on average. The two flanking 100-bp sequences
are segments of DNA that were originally located approximately 3 kb apart in the genome of interest.
This method requires no cloning and generates 150,000 to 200,000 paired-end reads from a single Genome
Sequencer FLX instrument run with a total elapsed time – from genomic DNA sample preparation to
sequencing results – of less than four days.
This new protocol for generating a library of paired-end fragments can be used to determine the orientation
and relative positions of contigs produced by de novo shotgun sequencing and assembly. This 3K Long-Tag
Paired End protocol (Fig. 1) can also be used to identify genomic structural variations and their associated
breakpoints. Structural variation of the genome, involving large, kilo- to mega-base sized deletions,
duplications, insertions, inversions, and complex combinations of rearrangements, is widespread in humans
and presumably responsible for a considerable amount of phenotypic variation.1
Figure Legend: Description of the 3K Long-Tag Paired End Sequencing protocol. Genomic DNA is randomly fragmented and subsequently circularized via adaptors. After circularization, the DNA is randomly fragmented, followed by enrichment for
linker-positive fragments via streptavidin-bead (SA) capture. Long Paired End Adaptors (A and B; recipe provided in the GS FLX
Paired End DNA Library Preparation Method Manual) are then ligated onto the linker-positive fragment ends. Fragments that contain
the A and B Adaptors are captured via streptavidin beads (SA) for emPCR amplification and subsequent 454 SequencingTM.
REFERENCE
1. Korbel, J.O. et al. Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome. Science 318, 420-426 (2007).
7|
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Bibliography of peer reviewed articles using the
Genome Sequencer Platform
HIV Studies
Hoffmann, C. et al. “DNA bar coding and pyrosequencing
to identify rare HIV drug resistance mutations.”
Nucleic Acids Research 35 (13), e91 (2007).
Wang, C. et al. “Characterization of mutation spectra with
ultra-deep pyrosequencing: Application to HIV-1 drug
resistance.” Genome Research 17 (8), 1195-1201 (2007).
Wang, G.P. et al. “HIV integration site selection: Analysis
by massively parallel pyrosequencing reveals association
with epigenetic modifications.” Genome Research 17 (8),
1186-1194 (2007).
BACs/Plastids/Mitochondria/etc.
Baker, S. et al. “A novel linear plasmid mediates flagellar
variation in Salmonella Typhi.” PLoS Pathogens 3 (5), e59
(2007).
Cai, Z. et al. “Complete plastid genome sequences of
Drimys, Liriodendron, and Piper: implications for the
phylogenetic relationships of magnoliids.” BMC
Evolutionary Biology 6, 77 (2006).
Cuadros-Orellana, S. et al. “Genomic plasticity in
prokaryotes: the case of the square haloarchaeon.”
The ISME Journal 1 (3), 235–245 (2007).
Jex, A.R. et al. “Using 454 technology for long-PCR based
sequencing of the complete mitochondrial genome from
single Haemonchus contortus (Nematoda).”
BMC Genomics 9, 11 (2008).
Meyer, M. et al. “Targeted high-throughput sequencing
of tagged nucleic acid samples.” Nucleic Acids Research
35 (15), e97 (2007).
Moore, M.J. et al. “Rapid and accurate pyrosequencing of
angiosperm plastid genomes.” BMC Plant Biology 6, 17 (2006).
Wicker, T. et al. “454 sequencing put to the test using the
complex genome of barley.” BMC Genomics 7, 275 (2006).
View a selection of these publications and learn
more about the Genome Sequencer FLX System at
www.genome-sequencing.com
NEW
Metagenomics and Microbial Diversity
Angly, F.E. et al. “The marine viromes of four oceanic
regions.” PLoS Biology, 4 (11), e368 (2006).
Cox-Foster, D.L. et al. “A metagenomic survey of
microbes in honey bee colony collapse disorder.”
Science 318 (5848), 283-287 (2007).
NEW Desnues C. et al. “Biodiversity and biogeography
of phages in modern stromatolites and thrombolites.”
Nature, 2008 Mar 2 [Epub ahead of print].
NEW Dinsdale E. A. et al. “Microbial ecology of four coral
atolls in the northern line islands.” PLoS ONE, 3 (2):e1584
(2008).
NEW Dinsdale E. A. et al. “Functional metagenomic profiling
of nine biomes.” Nature, 2008 Mar 12 [Epub ahead of print]
NEW Dowd S. E. et al. “Survey of bacterial diversity in
chronic wounds using Pyrosequencing, DGGE, and full
ribosome shotgun sequencing.” BMC Microbiology, 8 (1),
43 (2008).
Edwards, R.A. et al. “Using pyrosequencing to shed light on
deep mine microbial ecology.” BMC Genomics 7, 57 (2006).
Huber, J.A. et al. “Microbial population structures in the deep
marine biosphere.” Science 318 (5847), 97-100 (2007).
Huson, D.H. et al. “MEGAN analysis of metagenomic
data.” Genome Research 17 (3), 377-386 (2007).
NEW Jorge Frias-Lopez, J. et al. “Microbial community
gene expression in ocean surface waters.” Proc Natl.
Acad. Sci. USA, 105 (10), 3805–3810 (2008).
NEW Krause, L. et al. “Phylogenetic classification of
short environmental DNA fragments.” Nucleic Acids
Research, (February 19, 2008) [Epub ahead of print].
Krause, L. et al. “Finding novel genes in bacterial
communities isolated from the environment.”
Bioinformatics 22 (14), e281-e289 (2006).
Leininger, S. et al. “Archaea predominate among
ammonia-oxidizing prokaryotes in soils.” Nature 442
(7104), 806-809 (2006).
NEW Luiz F. W. et al. “Pyrosequencing enumerates and
contrasts soil microbial diversity.” The ISME Journal, 1,
283–290 (2007).
indicates journal articles that have been published since February 12, 2008.
NEW McKenna P. et al. “The Macaque Gut Microbiome
in Health, Lentiviral Infection, and Chronic
Enterocolitis.” PLoS Pathogens, 4 (2), e20 (2008).
NEW Mulholland P. J. et al. “Stream denitrification
across biomes and its response to anthropogenic
nitrate loading.” Nature 452, 202-205 (2008).
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Metagenomics and Microbial Diversity (continued)
Palacios, G. et al. “A new arenavirus in a cluster of fatal
transplant-associated diseases.” The New England Journal
of Medicine 358; published at www.nejm.org on February 6,
2008; doi:10.1056/NEJMoa073785 (2008).
Ancient DNA
Sogin, M.L. et al. “Microbial diversity in the deep sea and
the underexplored ‘rare biosphere’.” Proc. Natl. Acad. Sci.
USA 103 (32), 12115-12120 (2006).
Briggs, A.W. et al. “Patterns of damage in genomic DNA
sequences from a Neandertal.” Proc. Natl. Acad. Sci. USA
104 (37), 14616-14621 (2007).
Sundquist, A. et al. “Bacterial flora typing with deep,
targeted, chip-based Pyrosequencing.”
BMC Microbiology 7, 108 (2007).
Gilbert, M.T. et al. “Recharacterization of ancient DNA
miscoding lesions: insights in the era of sequencingby-synthesis.” Nucleic Acids Research 35 (1), 1-10 (2007).
Turnbaugh, P.J. et al. “An obesity-associated gut microbiome with increased capacity for energy harvest.”
Nature 444 (7122), 1027-1031 (2006).
Gilbert, M.T. et al. “Whole-genome shotgun sequencing
of mitochondria from ancient hair shafts.” Science 317
(5846), 1927-1930 (2007).
Warnecke, F. et al. “Metagenomic and functional analysis
of hindgut microbiota of a wood-feeding higher termite.”
Nature 450 (7169), 560-565 (2007).
Green, R.E. et al. “Analysis of one million base pairs of
Neanderthal DNA.” Nature, 444 (7117), 330-336 (2006).
Wegley, L. et al. “Metagenomic analysis of the microbial
community associated with the coral Porites astreoides.”
Environmental Microbiology 9 (11), 2707-2719 (2007).
ChIP Sequencing/Epigentics/
Structural Variation
Albert, I. et al. “Translational and rotational settings of
H2A.Z nucleosomes across the Saccharomyces
cerevisiae genome.” Nature 446 (7135), 572-576 (2007).
Bhinge, A.A. et al. “Mapping the chromosomal targets of
STAT1 by Sequence Tag Analysis of Genomic Enrichment
(STAGE).” Genome Research 17 (6), 910-916 (2007).
Boyle, A.P. et al. “High-resolution mapping and characterization of open chromatin across the genome.” Cell
132 (2), 311-322 (2008).
NEW Chen, J. et al. “Scanning the human genome at
kilobase resolution.” Genome Research (published online
Feb 21, 2008).
Dostie, J. et al. “Chromosome conformation capture
carbon copy (5C): a massively parallel solution for
mapping interactions between genomic elements.”
Genome Research 16 (10), 1299-1309 (2006).
Johnson, S.M. et al. “Flexibility and constraint in the
nucleosome core landscape of Caenorhabditis elegans
chromatin.” Genome Research 16 (12), 1505-1516 (2006).
Korbel, J.O. et al. “Paired-end mapping reveals extensive
structural variation in the human genome.” Science 318
(5849), 420-426 (2007).
Nagel, S. et al. “Activation of TLX3 and NKX2-5 in t(5;14)
(q35;q32) T-Cell acute lymphoblastic leukemia by
remote 3'-BCL11B enhancers and coregulation by PU.1
and HMGA1.” Cancer Research 67 (4), 1461-1471 (2007).
9|
Swaminathan, K., Varala, K., and Hudson, M.E. “Global
repeat discovery and estimation of genomic copy
number in a large, complex genome using a highthroughput 454 sequence survey.” BMC Genomics 8,
132 (2007).
Noonan, J.P. et al. “Sequencing and analysis of
Neanderthal genomic DNA.” Science 314 (5802), 11131118 (2006).
Poinar, H.N. et al. “Metagenomics to paleogenomics:
large-scale sequencing of mammoth DNA.” Science 311
(5759), 392-394 (2006).
Stiller, M. et al. “Patterns of nucleotide misincorporations
during enzymatic amplification and direct large-scale
sequencing of ancient DNA.” Proc. Natl. Acad. Sci. USA
103 (37), 13578-13584 (2006).
Whole Genome Sequencing
Andries, K. et al. “A diarylquinoline drug active on the
ATP synthase of Mycobacterium tuberculosis.” Science
307 (5707), 223-227 (2005).
NEW Bekal S. et al. “Genomic DNA sequence comparison between two inbred soybean cyst nematode biotypes facilitated by massively parallel 454 micro-bead
sequencing.” Mol Genet Genomics, 2008 Mar 7 [Epub
ahead of print].
Chen, Y. et al. “The capsule polysaccharide structure and
biogenesis for non-O1 Vibrio cholerae NRT36S: genes
are embedded in the LPS region.” BMC Microbiology 7,
20 (2007).
Chen, Y. et al. “The genome of non-O1 Vibrio cholerae
NRT36S demonstrates the presence of pathogenic
mechanisms that are distinct from O1 Vibrio cholerae.”
Infection and Immununity, 75 (5), 2645-2647 (2007).
Deng, L. et al. “Identification of novel antipoxviral
agents: mitoxantrone inhibits vaccinia virus replication
by blocking virion assembly.” Journal of Virology 81 (24),
13392-13402 (2007).
NEW Giannakis M. et al. “Helicobacter pylori evolution
during progression from chronic atrophic gastritis to
gastric cancer and its impact on gastric stem cells.”
Proc Natl. Acad. Sci. USA, 105 (11), 4358-4363 (2008).
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Whole Genome Sequencing (continued)
Gioia, J. et al. “Paradoxical DNA repair and peroxide
resistance gene conservation in Bacillus pumilus SAFR032.” PLoS ONE 2 (9), e928 (2007).
Goldberg, S.M. et al. “A Sanger/pyrosequencing hybrid
approach for the generation of high-quality draft assemblies of marine microbial genomes.” Proc. Natl. Acad. Sci.
USA 103 (30), 11240-11245 (2006).
Hallen, H.E. et al. “Gene family encoding the major
toxins of lethal Amanita mushrooms.” Proc. Natl. Acad.
Sci. USA 104 (48), 19097-19101 (2007).
Highlander, S.K. et al. “Subtle genetic changes enhance
virulence of methicillin resistant and sensitive
Staphylococcus aureus.” BMC Microbiology 7, 99 (2007).
Hiller, N.L. et al. “Comparative genomic analyses of
seventeen Streptococcus pneumoniae strains: insights
into the pneumococcal supragenome.” Journal of
Bacteriology 189 (22), 8186-8195 (2007).
Hofreuter, D. et al. “Unique features of a highly pathogenic Campylobacter jejuni strain.” Infection and Immunity
74 (8), 4694-4707 (2006).
Hogg, J.S. et al. “Characterization and modeling of the
Haemophilus influenzae core- and supra-genomes
based on the complete genomic sequences of Rd and
12 clinical nontypeable strains.” Genome Biology 8 (6),
R103 (2007).
NEW Hongoh, Y. et al. “Complete genome of the uncultured Termite Group 1 bacteria in a single host protist
cell.” Proc Natl. Acad. Sci. USA, 105 (14), 5555-5560 (2008)
Lasken, R.S. and Stockwell, T.B. “Mechanism of chimera
formation during the Multiple Displacement
Amplification Reaction.” BMC Biotechnology 7, 19 (2007).
NEW La Scola, B. et al. “Rapid comparative genomic
analysis for clinical microbiology: The Francisella
tularensis paradigm.” Genome Research, published online
Apr 11, 2008
NEW Lee, J.S. et al. “Mutation in the Transcriptional
Regulator PhoP Contributes to Avirulence of
Mycobacterium tuberculosis H37Ra Strain.” Cell Host &
Microbe, 3, 97–103 (2008).
Page 11
DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and
comparison to soybean and Medicago truncatula.”
BMC Genomics 8, 427 (2007).
Malhi, R.S. et al. “MamuSNP: a resource for rhesus
macaque (Macaca mulatta) genomics.” PLOS One 2 (5),
e438 (2007).
McCutcheon, J.P. and Moran, N.A. “Parallel genomic evolution and metabolic interdependence in an ancient
symbiosis.” Proc. Natl. Acad. Sci. USA 104 (49),
19392–19397 (2007).
Mouton, L. et al. “Variable-number tandem repeats as
molecular markers for biotypes of Pasteuria ramosa in
Daphnia spp.” Applied and Environmental Microbiology 73
(11), 3715–3718 (2007).
Mußmann, M. et al. “Insights into the genome of large
sulfur bacteria revealed by analysis of single filaments.”
PLoS Biology 5 (9), e230 (2007).
Oh, J.D. et al. “The complete genome sequence of a
chronic atrophic gastritis Helicobacter pylori strain:
evolution during disease progression.” Proc. Natl. Acad.
Sci. USA 103 (26), 9999-10004 (2006).
Pearson, B.M. et al. “The complete genome sequence of
Campylobacter jejuni strain 81116 (NCTC11828).”
Journal of Bacteriology 189 (22), 8402-8403 (2007).
Pinard, R. et al. “Assessment of whole genome
amplification-induced bias through high-throughput,
massively parallel whole genome sequencing.”
BMC Genomics 7, 216 (2006).
NEW Paustian, M.L. et al. “Comparative genomic analysis of Mycobacterium avium subspecies obtained from
multiple host species.” BMC Genomics, 9 (1), 135 (2008).
Pol, A. et al. “Methanotrophy below pH 1 by a new
Verrucomicrobia species.” Nature 450 (7171), 874-878
(2007).
Poly, F. et al. “Genome sequence of a clinical isolate of
Campylobacter jejuni from Thailand.” Infection and
Immunity 75 (7), 3425-3433 (2007).
Raymond, J.A., Fritsen, C., and Shen, K. “An ice-binding
protein from an Antarctic sea ice bacterium.” FEMS
Microbiology Ecology 61 (2), 214-221 (2007).
Macas, J., Neumann, P., and Navratilova, A. “Repetitive
Flowgram showing a single read of 256 bases.
Each bar represents a discrete base (A, T, C, or G),
and the height of a bar correlates to the number of
bases in a specific position.
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Page 12
Whole Genome Sequencing (continued)
Smith, M.G. et al. “New insights into Acinetobacter
baumannii pathogenesis revealed by high-density
pyrosequencing and transposon mutagenesis.”
Genes & Development 21 (5), 614 (2007).
NEW Solda, G. et al. “Non-random retention of
protein-coding overlapping genes in Metazoa.” BMC
Genomics, 9(1), 174 (2008).
NEW Stephen J. Spatz, S.J. and Rue, C. A, “Sequence
determination of a mildly virulent strain (CU-2) of
Gallid herpesvirus type 2 using 454 pyrosequencing.”
Virus Genes, DOI 10.1007/s11262-008-0213-5 (2008).
Swingley, W.D. et al. “Niche adaptation and genome
expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina.” Proc. Natl. Acad. Sci. USA 105
(6), 2005-2010 (2008).
NEW Tauch, A. et al. “The lifestyle of Corynebacterium
urealyticum derived from its complete genome
sequence established by pyrosequencing.” Journal of
Biotechnology, Available online 10 March 2008.
NEW Tauch, A. et al. “Ultrafast pyrosequencing of
Corynebacterium kroppenstedtii DSM44385 revealed
insights into the physiology of a lipophilic corynebacterium that lacks mycolic acids.” Journal of Biotechnology,
Available online 20 March 2008
Thomson, N.R. et al. “Chlamydia trachomatis: genome
sequence analysis of lymphogranuloma venereum
isolates.” Genome Research 18 (1), 161-171 (2008).
Velicer, G.J. et al. “Comprehensive mutation identification
in an evolved bacterial cooperator and its cheating
ancestor.” Proc. Natl. Acad. Sci. USA 103 (21), 8107-8112
(2006).
Velasco, R. et al. “A high quality draft consensus
sequence of the genome of a heterozygous grapevine
variety.” PLoS One 2 (12), e1326 (2007).
Wackett, L.P. et al. “Genomic and biochemical studies
demonstrating the absence of an alkane-producing
phenotype in Vibrio furnissii M1.” Applied and
Environmental Microbiology 73 (22), 7192-7198 (2007).
NEW Wheeler, D.A. et al. “The complete genome of an
individual by massively parallel DNA sequencing.”
Nature 452, 872 - 876 (2008).
Zuber, S. et al. “Genome analysis of phage JS98 defines
a fourth major subgroup of T4-like phages in Escherichia
coli.” Journal of Bacteriology 189 (22), 8206-8214 (2007).
View a selection of these publications and learn
more about the Genome Sequencer FLX System at
www.genome-sequencing.com
NEW
Thomas, J.A. et al. “Complete genomic sequence and
mass spectrometric analysis of highly diverse, atypical
Bacillus thuringiensis phage 0305varphi8-36.”
Virology 368 (2), 405-421 (2007).
11 |
indicates journal articles that have been published since February 12, 2008.
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Transcriptomes Sequencing
Bainbridge, M.N. et al. “Analysis of the prostate cancer
cell line LNCaP transcriptome using a sequencing-bysynthesis approach.” BMC Genomics 7, 246 (2006).
Barbazuk, W.B. et al. “SNP discovery via 454 transcriptome
sequencing.” The Plant Journal 51 (5), 910-918 (2007).
Cheung, F. et al. “Sequencing Medicago truncatula
expressed sequenced tags using 454 Life Sciences
technology.” BMC Genomics 7, 272 (2006).
Emrich, S.J. et al. “Gene discovery and annotation using
LCM-454 transcriptome sequencing.” Genome Research
17 (1), 69-73 (2007).
Eveland, A.L., McCarty, D.R., and Koch, K.E. “Transcript
profiling by 3'-untranslated region sequencing resolves
expression of gene families.” Plant Physiology 146 (1),
32-44 (2008).
Feng, H. et al. “Clonal integration of a polyomavirus in
human Merkel cell carcinoma.” Science Published online
January 17, 2008; doi:10.1126/science.1152586 (2008).
Gowda, M. et al. “Robust analysis of 5'-transcript ends
(5'-RATE): a novel technique for transcriptome analysis
and genome annotation.” Nucleic Acids Research 34 (19),
e126 (2006).
Jones-Rhoades M.W., Borevitz J.O., and Preuss, D.
“Genome-wide expression profiling of the Arabidopsis
female gametophyte identifies families of small,
secreted proteins.” PLoS Genetics 3 (10), e171 (2007).
Ng, P. et al. “Multiplex sequencing of paired-end ditags
(MS-PET): a strategy for the ultra-high-throughput
analysis of transcriptomes and genomes.” Nucleic Acids
Research 34 (12), e84 (2006).
Nielsen, K.L. et al. “DeepSAGE-digital transcriptomics
with high sensitivity, simple experimental protocol and
multiplexing of samples.” Nucleic Acids Research 34 (19),
e133 (2006).
Ohtsu, K. et al. “Global gene expression analysis of
the shoot apical meristem of maize.” (Zea mays L.).
The Plant Journal 52 (3), 391-404 (2007).
Peano, C. et al. “Complete gene expression profiling of
Saccharopolyspora erythraea using GeneChip DNA
microarrays.” Microbial Cell Factories 6 (1), 37; Published
online November 26, 2007; doi:10.1186/1475-2859-6-37 (2007).
Robb, S.M., Ross, E., and Alvarado, A.S. “SmedGD: the
Schmidtea mediterranea genome database.” Nucleic
Acids Research 36 (Database issue), D599-D606 (2008).
Ruan, Y. et al. “Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive
transcriptome analysis using Paired-End diTags (PETs).”
Genome Research 17 (6), 828-838 (2007).
Page 13
Torres, T.T. et al. “Gene expression profiling by massively
parallel sequencing.” Genome Research 18 (1), 172-177
(2008).
Toth, A.L. et al. “Wasp gene expression supports
an evolutionary link between maternal behavior and
eusociality.” Science 318 (5849), 441-444 (2007).
Vera, J.C. et al. “Rapid transcriptome
characterization for a nonmodel organism using
454 pyrosequencing.” Molecular Ecology (OnlineEarly
Articles) Published online February 5, 2008;
doi:10.1111/j.1365-294X.2008.03666.x (2008).
NEW Wang, X. et al. “Using RNase sequence specificity
to refine the identification of RNA-protein binding
regions.” BMC Genomics, 9 (Suppl 1), S17 (2008).
Weber, A.P.M. et al. “Sampling the Arabidopsis
transcriptome with massively-parallel pyrosequencing.”
Plant Physiology 144 (1), 32-42 (2007).
Amplicons and Methylation
Binladen, J. et al. “The use of coded PCR primers
enables high-throughput sequencing of multiple
homolog amplification products by 454 parallel
sequencing.” PLoS ONE 2 (2), e197 (2007).
Dahl, F. et al. “Multigene amplification and massively
parallel sequencing for cancer mutation discovery.”
Proc. Natl. Acad. Sci. USA 104 (22), 9387-9392 (2007).
Korshunova, Y. et al. “Massively parallel bisulphite
pyrosequencing reveals the molecular complexity
of breast cancer-associated cytosine-methylation
patterns obtained from tissue and serum DNA.”
Genome Research 18 (1), 19-29 (2008).
Marshall, H.M. et al. “Role of PSIP1/LEDGF/p75
in lentiviral infectivity and integration targeting.”
PLoS ONE 2 (12), e1340 (2007).
Ordway, J.M. et al. “Identification of novel highfrequency DNA methylation changes in breast cancer.”
PLoS ONE 2 (12), e1314 (2007).
Pettersson, E. et al. “Allelotyping by massively parallel
pyrosequencing of SNP-carrying trinucleotide threads.”
Human Mutation 29 (2), 323-329 (2008).
Taylor, K.H. et al. “Ultradeep bisulfite sequencing
analysis of DNA methylation patterns in multiple gene
promoters by 454 sequencing.” Cancer Research 67 (18),
8511-8518 (2007).
Thomas, R.K. et al. “Sensitive mutation detection in
heterogeneous cancer specimens by massively parallel
picoliter reactor sequencing.” Nature Medicine 12 (7),
852-855 (2006).
Thomas, R.K. et al. “High-throughput oncogene mutation
profiling in human cancer.” Nature Genetics 39 (3), 347351 (2007).
NEW Sugarbaker, D. J., et al. “Transcriptome sequencing
of malignant pleural mesothelioma tumors.” Proc Natl.
Acad. Sci. USA, 105 (9), 3521–3526 (2008).
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Technology and Bioinformatics
Brockman, W. et al. “Quality scores and SNP detection in
sequencing-by-synthesis systems.”
Genome Research Published online ahead of print
January 22, 2008 as doi:10.1101/gr.070227.107 (2008).
Chaisson, M.J. and Pevzner, P.A. “Short read
fragment assembly of bacterial genomes.”
Genome Research 18 (2), 324-330 (2008).
NEW Hamady, M. et al. “Error-correcting barcoded
primers for pyrosequencing hundreds of samples in
multiplex.” Nature Methods 5, 235 - 237 (2008).
Huse, S.M. et al. “Accuracy and quality of massivelyparallel DNA pyrosequencing.” Genome Biology 8 (7),
R143 (2007).
Liu, Z. et al. “Short pyrosequencing reads suffice for
accurate microbial community analysis.” Nucleic Acids
Research 35 (18), e120 (2007).
Marcy, Y. et al. “Nanoliter reactors improve multiple
displacement amplification of genomes from single
cells.” PLoS Genetics 3 (9), e155 (2007).
Margulies, M. et al. “Genome sequencing in microfabricated high-density picolitre reactors.” Nature 437
(7057), 376-380 (2005).
NEW Meyer, M., Stenzel, U., and Hofreiter, M. “Parallel
tagged sequencing on the 454 platform” Nature
Protocols 3, 267-278 (2008).
Meyer, M. et al. “From micrograms to picograms:
quantitative PCR reduces the material demands of
high-throughput sequencing.” Nucleic Acids Research 36
(1), e5 (2008).
Sequence Capture Arrays
(Sample Enrichment)
Albert, T.J. et al. “Direct selection of human genomic loci
by microarray hybridization.” Nature Methods 4 (11), 903905 (2007).
Small RNA
Aravin, A.A. et al. “Developmentally regulated piRNA
clusters implicate MILI in transposon control.” Science
316 (5825), 744-747 (2007).
Axtell, M.J. et al. “A two-hit trigger for siRNA biogenesis
in plants.” Cell 127 (3), 565–577 (2006).
Axtell, M.J., Snyder, J.A., and Bartel, D.P. “Common
functions for diverse small RNAs of land plants.”
Plant Cell 19 (6), 1750-1769 (2007).
Barakat, A. et al. “Conservation and divergence of
microRNAs in Populus.” BMC Genomics 8, 481 (2007).
Barakat, A. et al. “Large-scale identification of microRNAs
from a basal eudicot (Eschscholzia californica) and
conservation in flowering plants.” The Plant Journal 51
(6), 991-1003 (2007).
Berezikov, E. et al. “Diversity of microRNAs in human
and chimpanzee brain.” Nature Genetics 38 (12),
1375-1377 (2006).
Brennecke, J. et al. “Discrete small RNA-generating loci
as master regulators of transposon activity in
Drosophila.” Cell 128 (6), 1089 -1103 (2007).
Burnside, J. et al. “Marek’s disease virus encodes
microRNAs that map to meq and the latency-associated
transcript.” Journal of Virology 80 (17), 8778-8786 (2006).
Parameswaran, P. et al. “A pyrosequencing-tailored
nucleotide barcode design unveils opportunities for
large-scale sample multiplexing.” Nucleic Acids Research
35 (19), e130 (2007).
Calabrese, J.M. et al. “RNA sequence analysis defines
Dicer's role in mouse embryonic stem cells.” Proc. Natl.
Acad. Sci. USA 104 (46), 18097-18102 (2007).
Quinlan, A.R. et al. “Pyrobayes: an improved base caller
for SNP discovery in pyrosequences.” Nature Methods 5
(2), 179-181 (2008).
Fahlgren, N. et al. “High-throughput sequencing of
Arabidopsis microRNAs: evidence for frequent birth and
death of MIRNA genes.” PLoS ONE 2 (2), e219 (2007).
Trombetti, G.A. et al. “Data handling strategies for high
throughput pyrosequencers.” BMC Bioinformatics 8
(Suppl 1), S22 (2007).
Girard, A. et al. “A germline-specific class of small RNAs
binds mammalian Piwi proteins.” Nature 442 (7099),
199-202 (2006).
NEW Vacic, V. et al. “A probabilistic method for small
RNA flowgram matching.” Pacific Symposium on
Biocomputing; 75-86 (2008).
Henderson, I.R. et al. “Dissecting Arabidopsis thaliana
DICER function in small RNA processing, gene silencing
and DNA methylation patterning.” Nature Genetics 38 (6),
721-725 (2006).
van Orsouw, N.J. et al. “Complexity Reduction of
Polymorphic Sequences (CRoPSTM): a novel approach
for large-scale polymorphism discovery in complex
genomes.” PLoS ONE 2 (11), e1172 (2007).
Houwing, S. et al. “A role for Piwi and piRNAs in germ
cell maintenance and transposon silencing in
zebrafish.” Cell 129 (1), 69-82 (2007).
Howell, M.D. et al. “Genome-wide analysis of the
RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4
pathway in Arabidopsis reveals dependency on
miRNA- and tasiRNA-directed targeting.” Plant Cell 19
(3), 926-942 (2007).
13 |
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Small RNA (continued)
Jin, H. et al. “Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis.”
BMC Molecular Biology 9 (1), 6 Published online ahead of
print January 14, 2008; doi:10.1186/1471-2199-9-6 (2008).
Johnson, C. et al. “CSRDB: a small RNA integrated database and browser resource for cereals.” Nucleic Acids
Research 35 (Database issue), D829-D833 (2007).
Kasschau, K.D. et al. “Genome-wide profiling and analysis
of Arabidopsis siRNAs.” PLoS Biology 5 (3), e57 (2007).
Katiyar-Agarwal, S. et al. “A novel class of bacteriainduced small RNAs in Arabidopsis.” Genes &
Development 21 (23), 3123-3134 (2007).
Lau, N.C. et al. “Characterization of the piRNA complex
from rat testes.” Science 313 (5785), 363-367 (2006).
NEW Lu, J. et al. “The birth and death of microRNA
genes in Drosophila.” Nature Genetics, 40 (3), 351-355
(2008).
Lu, C. et al. “MicroRNAs and other small RNAs enriched
in the Arabidopsis RNA-dependent RNA polymerase-2
mutant.” Genome Research 16 (10), 1276-1288 (2006).
Molnár, A. et al. “miRNAs control gene expression in the
single-cell alga Chlamydomonas reinhardtii.” Nature 447
(7148), 1126-1129 (2007).
NEW Mosher RA, et al. “PolIVb influences RNA-directed
DNA methylation independently of its role in siRNA
biogenesis.” Proc Natl. Acad. Sci. USA,105 (8), 3145-3150
(2008).
Pak, J. and Fire, A. “Distinct populations of primary and
secondary effectors during RNAi in C. elegans.” Science
315 (5809), 241-244 (2007).
Page 15
NEW Seitz, H., Ghildiyal, M., and Zamore, P. D. “Argonaute
loading improves the 5' precision of both MicroRNAs
and their miRNA strands in flies.” Current Biology, 18 (2),
147-51 (2008).
NEW Sunkar, R. et al. “Identification of novel and candidate miRNAs in rice by high throughput Sequencing.”
BMC Plant Biology, 8, 25 (2008).
Tarasov, V. et al. “Differential regulation of microRNAs
by p53 revealed by massively parallel sequencing.”
Cell Cycle 6 (13), 1586-1593 (2007).
Tyler, D.M. et al. “Functionally distinct regulatory RNAs
generated by bidirectional transcription and
processing of microRNA loci.” Genes & Development 22
(1), 26-36 (2008).
Yao, Y. et al. “Cloning and characterization of microRNAs
from wheat (Triticum aestivum L.).” Genome Biology 8 (6),
R96 (2007).
Zhang, X. et al. “Role of RNA polymerase IV in plant
small RNA metabolism.” Proc. Natl. Acad. Sci. USA 104
(11), 4536-4541 (2007).
Zhang, L. et al. “Systematic identification of C. elegans
miRISC proteins, miRNAs, and mRNA targets by their
interactions with GW182 proteins AIN-1 and AIN-2.”
Molecular Cell 28 (4), 598–613 (2007).
Zhao, T. et al. “A complex system of small RNAs in the
unicellular green alga Chlamydomonas reinhardtii.”
Genes & Development 21 (10), 1190-1203 (2007).
View a selection of these publications and learn
more about the Genome Sequencer FLX System at
www.genome-sequencing.com
NEW
indicates journal articles that have been published since February 12, 2008.
NEW Pandey, S.P. et al. “Herbivory-induced changes in
the small-RNA transcriptome and phytohormone
signaling in Nicotiana attenuate.” Proc Natl. Acad. Sci.
USA, 2008 Mar 13 [Epub ahead of print].
Qi, Y. et al. “Distinct catalytic and non-catalytic roles of
ARGONAUTE4 in RNA-directed DNA methylation.”
Nature 443 (7114), 1008-1112 (2006).
Rajagopalan, R. et al. “A diverse and evolutionarily fluid
set of microRNAs in Arabidopsis thaliana.” Genes &
Development 20 (24), 3407-3425 (2006).
Ruby, J.G. et al. “Evolution, biogenesis, expression, and
target predictions of a substantially expanded set of
Drosophila microRNAs.” Genome Research 17 (12),
1850-1864 (2007).
Ruby, J.G., Jan, C.H., and Bartel, D.P. “Intronic microRNA
precursors that bypass Drosha processing.” Nature 448
(7149), 83-86 (2007).
Ruby, J.G. et al. “Large-scale sequencing reveals
21U-RNAs and additional microRNAs and endogenous
siRNAs in C. elegans.” Cell 127 (6), 1193-1207 (2006).
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