cancermetabolismresearch · 2014-05-16 · integrates 3 components: • color calibration...
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Albert-L·szlÛ Barab·si,
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The Human
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Researchers studying rare variant biomarkers often find
themselves on the lookout for faint genetic signals against an
overwhelming background, sometimes as little as a single positive
in 100,000 negatives or more. Such a situation cries out for
polymerase chain reaction (PCR), but standard PCR wonít do.
These days, thereís digital PCR (dPCR). By discretizing those
100,000 molecules in a large number of individual reactions,
dPCR makes the rare positive surprisingly easy to find.
See the full story on page 212.
The Digital PCR Revolution
Life Science Technologies
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changesódistinguishing six and seven copies, for instanceóand limited
sensitivity.
The resulting uncertainty can complicate, for instance, data sharingóa
critical limitation when setting up multi-institutional trials, says Muneesh
Tewari, associate professor at the University of Michigan and formerly
associate member of the Fred Hutchinson Cancer Research Center
(FHCRC), who uses dPCR for microRNA biomarker discovery. ìReal-time
PCR just doesnít have the day-to-day precision, or sometimes even the pre-
cision within the same day that we would like,î he says.
dPCR circumvents these issues using a ìdivide-and-conquerî strategy. A
mixture of molecules is discretized or compartmentalized into a large num-
ber of reaction chambersóthe more the betterósuch that each chamber
has on average either one target nucleic acid or none. Following PCR am-
plification of each compartment, Poisson statistics can be used to convert
the count of positive signals into an absolute number.
Kinzler likens the process to Sanger sequencing. Sequencing a mixture
of 100 template molecules, one of which is mutant, in a single tube would
yield an electropherogram in which the dominant signal at the mutated
position would be the wild-type base. ìYou probably wouldnít even be able
to tell there was a little hump underneath [the peak] for the alternative base
read, because it represents too small a percentage of the molecules.î But
diluting the molecules and distributing them in multiple reactions would
produce 99 wild-type signals and one clear mutant óa digital, and abso-
lutely quantitative, result.
Digital PCR essentially lifts weak signals out of the noise, and research-
ers today are using that strategy for everything from detecting copy number
variations and circulating tumor DNA to HIV viral load monitoring and fe-
tal aneuploidy testing. Clinical applications, in particular, seem most likely
to benefit from the technology, assuming it can be made simple enough for
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MicroscopyóMay 2
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Upcoming Features
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Kenneth Kinzler, co-director of the
Ludwig Center at Johns Hop-
kins University, who (with col-
league Bert Vogelstein) first coined the
term ìdigital PCR,î says he and Vogelstein
developed the approach to better identify
rare cancer mutations. ìThe thought that
occurred to us was that the most sensitive
[detection] you can get is by looking at
single molecules,î Kinzler explains. ìWhen
you start with single molecules, [reactions]
are either 100% mutant or 100% wild type,
which makes the distinction of a tumor
from wild type much easier.î
Of course, seeing even very rare events
using just standard PCR should be possible.
After all, PCR excels at plucking needles
from molecular haystacks. In theory, given
the right primers and cycling conditions,
the reaction can copy a single piece of
template DNA into millions upon millions
of daughter strands, enough to clone, se-
quence, or detect on a gel. But because the
process isnít quantitative, researchers can-
not infer the DNA content of the starting
sample from the number of molecules pres-
ent at the end of the reaction.
qPCR addresses that problem by quan-
tifying the reaction as it runs. By charting
fluorescence intensity over time, research-
ers can compare, for instance, the relative
expression of a given gene from sample
to sample. But absolute quantification by
qPCR isnít straightforward. It generally
requires standard curves to convert abun-
dance into absolute concentrations, and
those concentrations can sometimes vary
day to day and across labs. qPCR also strug-
gles with detecting subtle copy-number
The Digital PCR RevolutionìLike finding a needle in a haystackî is an overused expression, but when it comes to some
biological scavenger hunts, it fits. Researchers studying rare variant biomarkers often find
themselves on the lookout for faint genetic signals against an overwhelming background,
sometimes as little as a single positive in 100,000 negatives or more. Such a situation cries
out for polymerase chain reaction (PCR), a technique uniquely capable of capturing the
proverbial needle. But standard PCR wonít doóit is a qualitative techniqueóand neither
will quantitative real-time PCR (qPCR), which often lacks the necessary accuracy and
sensitivity. These days, thereís a third and increasingly popular option: digital PCR (dPCR).
By discretizing those 100,000 molecules in a large number of individual reactions, dPCR
makes the rare positive surprisingly easy to find. By Jeffrey M. Perkel
Digital PCR
essentially lifts
weak signals
out of the noise,
and researchers
today are using
that strategy for
everything from
detecting copy
number variations
and circulating
tumor DNA to
HIV viral load
monitoring.
Genomics
Life Science Technologies Produced by the Science��������������������� �����
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statistics describe a random distribution. As
long the compartments arenít saturated, us-
ers can back-calculate how many molecules
they started with, even if some wells actually
receive more than one molecule (an event
that, in dPCR, reads as a single count). ìYou
tell me how many compartments or how
many droplets you have, [and] what fraction
of those are negative. That will tell me es-
sentially what concentration I started with
that would give me that ratio of positive
compartments to negative compartments,î
he says.
Reginald Beer, medical diagnostics initiative leader at the Lawrence
Livermore National Laboratory, who as a graduate student published
the first report of dPCR in monodisperse (i.e., identically sized) droplets,
says the advantage of the droplet-based approach lies in its scalability: Itís
relatively simple to increase the number of reaction vessels, which in turn
increases data quality. ìPoisson accuracy greatly improves as you scale or
increase the number of your reactor vessels,î he explains.
Published reports establish the efficacy of both Bio-Radís and Rain-
Danceís dPCR platforms as well as the diversity of applications amenable to
dPCR. RainDanceís platform, for instance, has been used to detect mutant
gene transcripts in cerebrospinal fluid from glioma patients and KRAS mu-
tations in sera from colorectal cancer patients.
Jason Bielas, associate member of the FHCRC, used Bio-Radís system
to quantify tumor-infiltrating lymphocytes in ovarian cancer, while Keith
Jerome, professor and head of the Virology Division in the Department
of Laboratory Medicine at the University of Washington and associ-
ate member of the FHCRC, used it to develop a way to distinguish active
HHV-6 viremia from latent, genome-integrated virus. (The former must be
treated if it occurs during the course of bone marrow transplantation, but
not the latter; the two are distinguished by the ratio of viral sequence copies
to genome equivalents in blood, which in the case of genome integration
should be 1.0.)
Bielas says his team was able to use their own method, termed QuanTILfy,
to reproducibly resolve as little as one T lymphocyte in 10,000 cancer
cellsóenough to detect an association between T-cell tumor infiltration and
patient survival. That method is considerably more quantitative, he explains,
than the current standard of immunohistochemistry. ìIn
THE ORIGINAL DIGITAL PCR
Published in 1999, Kinzler and Vogelsteinís original ìdigital PCRî approach
was a tedious, manual affair. Looking to detect and quantify K-RAS
mutations in stool samples from colorectal cancer patients, the pair diluted
and aliquoted genomic DNA into each well of a 384-well microtiter plate,
such that the entire plate represented a single sample. They then amplified
the gene region that encompassed the mutational hotspots they were
looking for.
Once the reactions came to completionódPCR generally is an end-
point PCR assay, though it doesnít have to beóthey hybridized two fluores-
cent probes, a control that should always hybridize and a second that only
binds to wild-type sequences, and read the results. Just over 100 wells had
genomic DNA, of which four appeared to be mutants.
The results, Kinzler says, indicated dPCR was ìa very robust, reliable,
and accurateî method for rare mutation detection. But it also was labor-
intensive and difficult to scale. ìIt was often impossible to convince a grad
student to do it,î he says.
In 2003, Kinzler and Vogelstein described an improved protocol, which
they called ìBEAMingî for its reliance on ìbeads, emulsion, amplification,
and magnetics.î In place of manual sample discretizing in a microtiter plate,
BEAMing turns the individual droplets of a water-in-oil emulsion into re-
action vessels. Using an emulsion PCR process thatís now used widely in
next generation DNA sequence library preparation, BEAMing distributes
a mixture of template, primers, PCR reagents, and magnetic beads into
droplets, again under conditions such that most droplets will contain either
zero or one template. Following PCR, during which the amplified prod-
uct is coupled to the bead via a biotin-streptavidin linkage, the emulsion is
broken and the beads read by hybridization with detection oligonucleotides
and flow cytometry.
Instead of manual separation on a plate, BEAMing ìcan do the equivalent
of 100,000 wells,î says Kinzler, who still uses the technology and (with
Vogelstein and other colleagues) cofounded Inostics to commercialize it.
(Inostics subsequently was acquired by Sysmex, and today Kinzler holds no
equity in the company.)
Droplet-based strategies have also been commercialized by Bio-Rad
Laboratories and RainDance Technologies. Instead of magnetic
beads, both companies discretize (or, as RainDance President and Chief Ex-
ecutive Officer S. Roopom Banerjee puts it, ìdropletizeî) the PCR mixture
into either 20,000 nanoliter-sized (Bio-Radís QX200) or 10 million pico-
liter-sized (RainDance RainDrop) droplets. Following PCR amplification,
the reaction results are read by flowing the droplets past a fluorescence
excitation source and detector, like a flow cytometer without the cells.
ìThink of it as like a digital camera,î says Banerjee, who says RainDanceís
RainDrop system was designed with single-button, ìApple-like simplicity.
You have a complicated picture with lots of colors, lots of diversity. What
weíre doing is basically pixelizing that picture down to miniscule-sized pix-
els and then we literally read out every single pixel in order to say exactly
whatís in that biological picture.î In RainDanceís case, it takes four hours to
read 80 million such ìpixels,î the output of eight parallel dPCR reactions.
POWERFUL POISSON
Because dPCR is an endpoint assay, the resulting concentration could in the-
ory be thrown off if the target molecules didnít discretize perfectlyóthat
is, some compartments ended up with more than one target. Thatís where
Poisson statistics come in. According to George Karlin-Neumann, director
of scientific affairs at Bio-Rad Laboratoriesí Digital Biology Center, Poisson
The technique
clearly offers
considerable
advantages for a
growing number
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Jeffrey M. Perkel is a freelance science writer based in Pocatello, Idaho.
www.sciencemag.org/products
essence, youíre counting T-cell genomes.î
Hanlee Ji, senior associate director of the Stanford Genome
Technology Center, also uses Bio-Rad, both to validate genetic
aberrations from genome-sequencing studies and more recently to quality
control next-gen libraries prior to sequencing. For his group, the advantage
of dPCR is largely simplicity: He can get an undergrad in the lab up to speed
on dPCR in weeks, whereas qPCR can take considerably longer.
ìOne of my tests of technology is: If I take an undergrad who has rea-
sonably good bench skills and sit them in front of an instrument, can they
generate robust results from the positive and negative controls that theyíre
asked to do within a matter of weeks? If so, that says a lot regarding how
well the system works.î
DIGITAL PCR-BY-SEQUENCING
In 2011, Kinzler and Vogelstein described yet another approach to dPCR
for rare allele detection, this time based on sequencing. Called Safe-
Sequencing System (Safe-SeqS), the strategy entails tagging individual
template molecules with unique identifiers (or barcodes), which are then
amplified and read out by next generation DNA sequencing.
According to Nickolas Papadopoulos, director of translational genetics
at the Ludwig Center at Johns Hopkins, professor of oncology at Johns
Hopkins Medical Institutes, and coauthor of the study: Safe-SeqS enables
researchers to extend the capacity of dPCR to many more locióup to 30
or moreóthan BEAMing can reasonably handle.
ìMassively parallel sequencing represents a particularly powerful form
of dPCR in that hundreds of millions of template molecules can be analyzed
one by one,î explain Papadopoulos and his coauthors in their 2011 study. ìIt
has the advantage over conventional dPCR methods in that multiple bases
can be queried sequentially and easily in an automated fashion.î But NGS
also has an unacceptably high inherent error rate caused by mistakes in am-
plification, sequencing, and detection. By tagging each starting molecule
with a unique identifier, Safe-SeqS enables researchers to differentiate true
mutations from procedural artifacts.
In the original study, the authors assessed mutations in the CTN-
NB1 gene in 100,000 normal human cells. Raw Illumina sequencing
reads produced an error rate 2.1x10-4. Taking into account the Safe-SeqS
barcodes lowered that rate 24-fold, to 9x10-6. When applied to mitochon-
drial DNA, Safe-SeqS dropped the observed mutation rate 15-fold.
This past year, the team extended Safe-SeqS to search for multiplexed
signatures of ovarian, endometrial, and cervical cancer in the scraped cervi-
cal material that comes from routine Pap testing, a first step in developing
an early detection system for gynecologic tumors. In this case, the team
used the assay to assess mutations in 46 gene regions from 12 cancer-asso-
ciated genes.
Papadopoulos, who discussed the method at a recent digital biology con-
ference in San Diego, says Safe-SeqS may be particularly useful for early
tumor detection from plasma. Overall, his team could detect mutations in
more than 80% of tumor plasma samples. But some tumor types worked
better than others. Particularly difficult, he says, were malignancies in
brain. ìCurrently, we can detect mutations in the plasma from only 10% of
the patients with these tumor types,î he says.
ARRAY-BASED STRATEGIES
For Andrzej Pietrzykowski, assistant professor of animal sciences at
Rutgers University, who studies the molecular and genetic bases of
alcoholism, dPCRís precision is particularly beneficial. Many of the changes
he sees are less than twofold and thus difficult to discern by qPCR. That
difference is readily detected digitally, he says.
But Pietrzykowski also cites another advantage. He has identified a par-
ticular microRNA that seems to be associated with alcoholism, and can
quantify that molecule using qPCR. But qPCR assays for microRNA pre-
cursors are harder to pull off, as identifying reliable controls for standard
curve calibration can be problematic. ìYou need to Ö have that housekeep-
ing gene double or triple-checked that it doesnít change due to conditions.î
Thatís a non-issue with dPCR, though, as the technique doesnít require a
standard curve.
Pietrzykowski was one of five Innovation Grant recipients of Life
Technologiesí Digital PCR Applications Grant program, which in late
2013 provided him with the companyís QuantStudio 3-D chip reader, chip
loader, and thermo cycler. The QuantStudio 3D Digital PCR system is like a
souped-up version of Kinzlerís original dPCR implementation. Researchers
use a tool akin to a windshield ìsqueegeeî to discretize samples onto 20,000
wells etched into the surface of a silicon wafer. Fluidigm, too, uses this
physical array strategy; its qdPCR 37K ìintegrated fluidic circuitî leverages
the companyís microfluidics expertise to distribute 48 samples into 770
chambers each (though a single sample can be spread over multiple sets of
chambers to increase accuracy, up to 37,000).
The technique clearly offers considerable advantages for a growing num-
ber of specific (and often clinical) applications. But donít discard your real-
time thermocycler just yet. For most researchers dPCR represents a com-
plement to qPCR, not a replacement. Indeed, for many applications, says
Beer, qPCR is ìby far sufficientîó plus, the technology is far more mature
and the assays established. ìqPCR certainly still has a significant advantage
from a throughput perspective,î says Iain Russell, senior product manager
for dPCR at Life Technologies (recently purchased by Thermo Fisher Scien-
tific), ìand to be quite honest, for a large number of applications out there
it meets all the needs of the customer.î
Says Pietrzykowski, ìIt really depends on what kind of question youíre
asking.î
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215
Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.dtl for more information.
Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are
featured in this space. Emphasis is given to purpose, chief characteristics, and availability of products and materials. Endorsement by Science or AAAS of any products or
materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.
DNA LIBRARY KIT
The Accel-NGS 2S DNA Library Kit for Illumina Next Generation Se-quencing (NGS) systems provides linear yields from inputs ranging from 10 pg to 1 µg. Polymerase chain reaction-free libraries can be produced from as little as 100 ng. Unlike other commercially available kits, exceptional quality and evenness of coverage are obtained even at very low levels of DNA input. The product is also is ideally suited for clinical samples such as FFPE tissues and plasma. Accel-NGS 2S enables NGS laboratories to stock and use a single kit for their varied DNA library needs, ranging from high molecular weight DNA to limited quantity, damaged FFPE samples. In ad-dition to whole genome sequencing, Accel-NGS 2S is also compatible with the leading hybridization capture products and is suitable for ChIP-Seq. Built with usability in mind, the 2S protocol is readily automatable for those with high throughput applications. The wide dynamic range of input makes pre-library quantification optional, further streamlining workflow.Swift Biosciences
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NGS LIBRARY PREP KITS
Current methods used for epigenetic analysis of DNA methylation are deficient in differentiating 5-methylcytosine from 5-hydroxymethylcyto-sine. The new Pico Methyl-Seq Library Prep and RRHP 5-hmC Library Prep kits are designed for NGS-based, whole-genome analysis of 5-meth-lycytosine and genome-wide analysis of 5-hydroxymethylcytosine in DNA, respectively. These unique products feature streamlined workflows and in-clude all the technologies required for consistent and robust library con-struction. Pico Methyl-Seq is a post-bisulfite library preparation method that can accommodate DNA inputs as low as 10 pg. Alternatively, RRHP allows for strand-specific, single-base profiling of 5-hmC from DNA in-puts as low as 100 ng by utilizing a bisulfite-free, enzymatic-based method. When combined, both technologies will elucidate genomic-scale methyla-tion and hydroxymethylation profiles for a particular DNA sample from any species. Zymo Research Corporation
For info: 888-882-9682 www.zymoresearch.com
POST-BISULFITE DNA LIBRARY PREPARATION
The new EpiNext Post-Bisulfite DNA Library Preparation Kit is designed for constructing DNA libraries directly from bisulfite-treated DNA for whole genome bisulfite sequencing. Bisulfite conversion is an essential pro-cess of modifying DNA so that methylated cytosine bases can be detected via sequencing. Currently used whole genome bisulfite sequencing meth-ods need rather large amounts of DNA (great than 1 µg) as input mate-rial, as well as requiring the need to first shear DNA and ligate adapters to DNA fragments prior to bisulfite conversion. The innovative technology developed by Epigentek allows bisulfite-treated DNA to be directly used for ligation, thereby eliminating the possibility of breaking adapter-ligated fragments, which can often occur in currently used next generation bisul-fite sequencing methods. The commercial kit, based on this technology, has high sensitivity and efficiency, enabling input DNA to be as low as 1 ng, and could be used for precious or limited biological samples.EpigentekFor info: 877-374-4368 www.epigentek.com
GENE-EDITING KITSThe new GENASSIST range of gene-editing kits and reagents enable easier, robust implementation of CRISPR and rAAV gene editing experiments. The current GENASSIST offering comprises both off-the-shelf reagents for using CRISPR editing technology and a unique kit combination of these reagents to allow customers to generate their own CRISPR-ready cell lines that constitutively express Cas9-nickase. Using such cell lines provides a quick start for customers, enabling them to make further modifications to the cell line more efficiently than if they were starting fresh each time. Ho-rizon is also launching a new service for the design, manufacture and most importantly validation of CRISPR RNA guides, in order to maximize the likelihood that gene editing will occur as expected. The new GENASSIST kits, combined with access to rAAV, ZFN, and CRISPR technologies, give researchers an invaluable suite of tools to determine the function of endog-enous gene alterations and their effect on disease and therapeutic responses. Horizon Discovery
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DESKTOP WHOLE-GENOME SEQUENCER
The NextSeq 500 System delivers the power of high throughput sequencing with the load-and-go simplicity of a desktop sequencer, effectively transforming a broad range of high throughput appli-cations into affordable, everyday research tools. Its push-button operation delivers a one-day turn-around for a number of popular sequencing applications, including one whole human genome and up to 16 exomes, up to 20 noninvasive prenatal testing samples, up to 20 transcriptomes, up to 48 gene expression samples, and up to 96 targeted panels. With its streamlined informatics, sequencing data can be run through a range of open -ource or commercial pipelines or instantly transferred, analyzed, and stored securely in BaseSpace or the new BaseSpace OnSite for researchers needing an on-premises solution. Users also have the flexibility to switch to lower throughput sequencing as needed, and while other platforms require several pieces of specialized equipment, the NextSeq 500 System integrates cluster generation and sequencing into a single instrument. Illumina For info: 800-809-4566 www.illumina.com/nextseq
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2.5
PhosphorylationofERK1/2
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No Drug PD 0325901 PD 98059 SL 327 U0126
Colon (COLO 205)
Breast (SK-BR-3)
Cervical (HeLa)
Breast (MDA-MB-468)
Melanoma (SK-Mel-28)
Epidermal (A431)
Lung (A549)