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Core Services
Mass Spectrometry
Luminex High Sensitivity Immunoassays
Flow Cytometry
HOMINGS (Human Oral Microbe Identification using
Next Generation Sequencing)
Biostructure and Imaging Core
Micro Computed Tomography (μCT) Core
Sequencing Core
Core capabilities as of February, 2015
Mass Spectrometry Core
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The Mass Spectrometry Core at the Forsyth Center for Salivary Diagnostics provides mass spectrometry
analysis and strategic consulting in proteomics for internal and external researchers. Three state-of-the-art mass
spectrometers are utilized to definitively identify and quantify proteins, peptides, metabolites and lipids that
may have diagnostic value. These instruments are interfaced with dedicated liquid chromatography and robotic
systems.
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AB Sciex 4800 Plus MALDI TOF/TOF
Thermo Scientific QExactive Plus
Thermo Scientific Orbitrap Fusion
Capabilities
Protein Identification (in gels or solution): the basic experimental workflow entails reduction/alkylation;
tryptic digestion, peptide extraction, nano-LC-MS/MS or MALDI-TOF MS/MS. The peptides are fragmented in
the mass spectrometer to yield diagnostic patterns that can be correlated to protein sequence databases via
computer algorithms for peptide identification. Proteins in complex solution backgrounds (e.g. detergent, nonvolatile salts, glycerol) require an additional cleanup-step to facilitate analysis.
Intact Molecular Weight Determination: Samples (intact protein, oligonucleotides, peptides) are applied to
stainless steel target place with appropriate matrix solution and air-dried. High sample concentration and
omission of salts and detergents are essential for efficient ionization.
Additional Services
Training, data interpretation and consulting, sample prep, and instrument operation are available at hourly rates
with advance notice to prepare time estimates and staff availability.
Instrument Access: AB Sciex 4800 MALDI-TOF/TOF Proteome Analyzer: The AB Sciex 4800 offers highly
accurate mass analysis (<10ppm) along with MS/MS capabilities for fragmentation analysis if peptides and
other biomolecules. Researchers can be trained for walk-up use.
Contact
Markus Hardt, Director: 617-892- 8350. mhardt@forsyth.org. http://proteomics.forsyth.org/
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Luminex Core
Bead-based flow cytometric multiplex arrays are useful for the detection of various biomarkers and proteins.
This technique uses microsphere beads, coated with monoclonal antibodies against specific proteins, to
simultaneously measure multiple analyte concentrations in various sample types including: body fluids
(serum, saliva, plasma, GCF, wound fluid, etc), cell extracts and culture supernatants. Luminex® and their
partners have developed many applications using the XMAP® Technology platform, including for the
following fields of life science research areas:
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Cancer Markers
Cardiovascular
Cell Signaling
Cellular Metabolism
Immunology
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Genotyping
Gene Expression Profiling
Endocrinology
Isotyping
Matrix Metalloproteinases
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Metabolic Endocrinology
Neurobiology
Transcription Factors
Toxicity
Forsyth has two platforms (Luminex® 200™ and FLEXMAP 3D™) which operate 96-well plate based assays.
FLEXMAP 3D™ also offers 384-well plate capacity. Plate capacity: up to 80 samples in singlet with up to 16
wells designated as standards for antigen-antibody based assays.
Capabilities
Multiplex protein detection using antibody-based assays of up to 100 analytes or 500 analytes in a single
microplate well using the Luminex® 200™ or FLEXMAP 3D™, respectively.
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Ability to custom coat beads with various mammalian and non-mammalian molecules such as nucleic
acids, receptor-ligands, and siRNA. These require custom-plexing, assay development/optimization, and
additional costs.
Both systems use BioPlex Manager from Bio-Rad, for acquisition and computation of data.
o Allows easy transfer of the data to Excel or other user-friendly programs.
o In addition to the raw and computed data on concentrations and fluorescence intensity, standard
curves, %CV, internal control samples, plate-to-plate variation, and standard deviation are only a
few of the data output measures.
Multiplex array results have compared similarly to measures from conventional techniques such as
ELISA. The cost/benefit ratio of this technology has also proven favorable over conventional methods in
terms of time, labor, cost, and particularly sample volume. While an ELISA may use up to 100 µl of
sample, multiplex technology requires only 5–25 μl of sample for multiple target detection, which
offers considerable advantages when limited research sample may be available.
Avoids the need for diluting samples multiple times or for multiple freeze-thawing of samples, each of
which can affect measurement accuracy and precision.
Cost effective compared to performing multiple ELISAs. The more analytes plexed together, the more
cost effective the assay.
Contact
Alpdogan Kantarci, Director: 617-892-8530 luminex@forsyth.org.
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Flow Cytometry Core
Flow Cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as
cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection
apparatus. It allows simultaneous multi-parametric analysis of the physical and/or chemical characteristics of up
to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders,
especially blood cancers, but has many other applications in both research and clinical practice. A common
variation is to physically sort particles based on their properties, so as to purify populations of interest. This
technique is known as fluorescence activated cell sorting (FACS), which uses fluorescent labels as a method for
detection.
Technology (The BD FACSAria™ II cell sorter)
The fluidics system in the BD FACSAria™ II cell sorter is pressure driven. Positive air pressure forces sample
cells through an optically gel-coupled cuvette flow cell. Hydrodynamic focusing guides particles in a single-file
stream through the cuvette, where laser light intercepts the stream at the sample interrogation point.
BD FACSAria II Laser and Filter Specifications (measures up to 13 colors simultaneously)
Laser Name
Red
Wavelength Filter
640
780/60 BP
730/45 BP
670/30 BP
Parameter
APC-Cy7
Alexa Fluor 700
APC
Blue
488
710/50 BP
530/30 BP
488/10 BP
PerCP-Cy5-5
FITC
SSC
Yellow Green
561
780/60 BP
660/20 BP
610/20 BP
582/15 BP
PE-Cy7
PE-Cy5
PE-Texas Red
PE
Violet
405
660/40 BP
605/40 BP
525/50 BP
450/50 BP
Qdot 655
Qdot 605
AmCyan
Pacific Blue
Contact
Daniel Nguyen, Manager: 617-892-8533. dnguyen@forsyth.org.
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HOMINGS
(Human Oral Microbe Identification using Next Generation Sequencing)
The latest generation utilizes the speed and efficiency of next generation sequencing combined with the
refinement of species-level identification.
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Species-level identification of nearly 600
oral bacterial taxa
Genus-level identification of remaining
sequences for 129 genera
High-throughput Illumina sequencing
In silico 16S rDNA probe analysis
Rapid—computationally-efficient
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More comprehensive than HOMIM
User-friendly dataset, e.g., % frequency
Traditional bioinformatics available, e.g.,
QIIME
Economical
At this time, HOMINGS are to be used only for research endeavors and cannot be used for diagnostic
purposes.
Analytic Capabilities
The focus of this service is to determine the oral human
microbiome; however, microbiomes from other human
body sites, e.g., intestinal, vaginal, skin, can also be
analyzed. Furthermore, microbiomes from animal or
environmental sources can also be determined. At
present, identification would be mostly at the genus or
higher taxonomic level. More information on our analytic
capabilities can be found at our website
(http://homings.forsyth.org)
Outputs are expressed in excel spreadsheets as %
frequencies of target taxa. If desired, arrangements can be
made to download raw data, e.g., FASTQ files. QIIME
analysis of raw data can be provided at an additional
charge, which is dependent upon the size and extent of
the project. These data will provide microbial profiles at
the genus or higher taxon level. For final analyses, %
frequency data can be incorporated in any number of
statistical packages, such as the R project, MeV, SAS,
and QIIME.
Facility and Instrumentation
This facility is staffed by 2 full-time and 3 part-time technicians who handle internal and external sequencing
needs. At present, we have 2 MiSeq (Illumina) bench top sequencers that can each generate up to 15 Gb of
output with 25 M sequencing reads and 2 X 250 bp read lengths. A NextSeq 500 (Illumina) has recently been
added to the facility, which has >10 times the capacity of the MiSeq. At present, 16S rDNA profiling is not
feasible on the NextSeq 500, but the instrument will eventually have the capacity to process at least 450
samples/run.
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Standard DNA screening and HOMINGS analysis. All samples will be initially screened to ensure that DNA is
of sufficient quality and quantity for sequence analysis. Samples deemed unsuitable will not be processed
further without consent from the investigator. MINIMUM ORDER is 10 samples. A purchase order or payment
plan is required prior to processing.
Additional Bioinformatic Analysis
Dependent upon time spent for analyses.
Contact
Dr. Bruce Paster, Director: 617-892-8288. bpaster@forsyth.org
Alexis Kokaras: 617-892-8567. akokaras@forsyth.org
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Biostructure and Imaging Core
This core facility provides consulting and support services associated with the preparation and analyses of both
soft tissues and mineralized tissue samples for histology and imaging analyses based on our expertise in all
steps of sample preparation including fixation, embedding, orienting, sectioning, immunohistochemistry, and
imaging. The core facility personnel, with 30 years of working experience, has a unique skill set for the
processing and orientations of mineralized tissue specimens.
Imaging Equipment
Zeiss Axio Observer Inverted Microscope with Zen Blue 2012 Image Acquisition software, Colibri.2 LED
light source for narrow band excitation with a reduction of cross stimulation and UV leak-through,
environmental chamber and Definite Focus for temperature and focus controlled time-lapse imaging;
Hamamatsu ORCA-Flash 4.0 - sCMOS camera with high quantum efficiency, low noise, 30fps at full
resolution; CRi Nuance fx Spectral Camera with sensitivity range 420nm-720nm, 20nm bandwidth for spectral
separating channels.
Zeiss LSM 780 Confocal Microscope with Zen Black 2012 software for acquisition, processing and analysis,
simultaneous detection for three channels, 32 Channel GaAsP detector for spectral analysis and separation,
linear unmixing and up to 45% quantum efficiency, environmental chamber and definite focus; Shuttle & Find
Software and sample holders for correlative microscopy in conjunction with Zeiss Evo LS10 SEM.
Zeiss Stemi SV11 Stereo microscope, great for larger specimens with Pentafluor and HBO light source for
fluorescence imaging, Axiocam HRc camera for monochrome or color imaging
Leica Upright light Microscopes:
Leica DMRE, Leica DMLS, and Leica DMR fluorescence microscope
JEOL 1200 EX transmission electron microscope for Bright/Darkfield imaging
Zeiss Evo LS10 scanning electron microscope for imaging in high vacuum and variable pressure mode for life
science application, equipped with Peltier stage for temperature control, backscattered electron detector and
Oxford INCA 250 microanalysis system for EDS
Workstation for Data Analysis
Dell Precision T7610 Computer with 64 GB RAM,
16- Core: Dual Intel Xeon Processor E5-2687W v2
(Eight Core HT, 3.4GHz Turbo, 25 MB) SSDs, 4
GB NVIDIA Quadro K5000 (490-BBKM), dual 27inch monitors
Software
Imaris, Confocal Standard Package, XT Programming Interface, MATLAB, Image
Processing Toolbox, Signal Processing Toolbox,
Parallel Computing Toolbox, Statistics Toolbox,
Computer Vision Toolbox
Contact
Dr. Felicitas Bidlack, Director: 617-892-8342. fbidlack@forsyth.org
Steven Wilbert: 617-892-8552. swilbert@forsyth.org
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Micro-Computed Tomography (μCT) Core
The Forsyth Micro Computed Tomography (μCT) Core offers micro 3D X-ray imaging of small samples in
high resolution, and will provide images and quantitative analyses of internal structures of ex vivo samples
without any destructive procedures. The Scanco μCT40 system allows for visualization, measurement, and
quantification of the structure of mineralized specimens. Contrast reagents permit visualization of structures
such as blood vessels. The non-destructive nature of this technology allows investigators to carry out
complementary analyses (e.g., histology) of the same samples.
Capabilities
The Core is equipped with a Scanco μCT 40, ex vivo μCT scanner, with a high throughput scanning option
(auto sample exchanger). This system creates a three-dimensional model from a stack of two-dimensional X-ray
images taken around a single axis of rotation. The high-resolution scanner can capture detail at six to 32
microns. The resolution is sample size-dependent. Scans taken of long bones have specific trabecular or
cortical analysis performed on them.
The system is equipped with automatic sample changer that allows high-throughput scanning. The sample
changer can hold maximum 10 sample tubes.
An additional software for advanced 3D analyses (Amira 3D Software for Life Sciences, FEI) will be
introduced in early 2015.
Links
Scanco μCT 40
http://www.scanco.ch/en/systems-solutions/specimen/microct40.html
Amira 3D Software for Life Sciences
http://www.fei.com/software/amira-3d-for-life-sciences/
Contact
Gen Sasaki, Director: 617-892-8459. hsasaki@forsyth.org.
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Sequencing Core
We can now quickly generate millions of high quality sequencing reads at very affordable prices. One of the
platforms at the forefront of this technology is Illumina, which employs the sequencing-by-synthesis (SBS)
chemistry, to track the addition of labelled nucleotides as the DNA chain is copied using a massively parallel
approach.
Facility and Instrumentation
The Sequencing core at the Forsyth Institute houses 2 MiSeqs (Illumina) and 1 Nextseq 500 (Illumina) to serve
the sequencing needs of researchers within and outside the Institute. The MiSeq is a bench top sequencer that
can generate up to 15 Gb of output with 25 M sequencing reads and up to 2 X 300 bp read lengths. It has the
highest accuracy per base as compared to other benchtop sequencers. The Nextseq 500 Sequencing system is
capable of delivering high-throughput, high quality sequencing with the speed and affordability of a desktop
sequencer. In about 30 hours, it can generate up to ~120 Gb of data with 400M reads at read lengths of 2x 150
bps. The simple workflow enables several sequencing applications including exomes, whole genomes and
transcriptomes for both mammalian and microbial models in a single run without the need for additional
standardized equipment.
Applications and Workflow
 16S rDNA profiling
 Microbial genome / Metagenome
 Microbial RNA / Metatranscriptome
 Mammalian genome
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Mammalian transcriptome
Targeted RNA expression studies
Amplicon based sequencing analysis for
eukaryotes.
Other applications can be developed as needed. Depending upon the queue, these in-house instruments provide
a rapid turn-around time as compared to typical outside sources.
The researcher provides the facility with good quality DNA or enriched mRNA depending on the service
requested. The samples are QC-ed using an Agilent Bioanalyzer before proceeding to library preparation. The
libraries are then QC-ed using both the bioanalyzer and Real time qPCR before being sequenced using the
appropriate instrument. Raw reads are supplied to the investigators.
Sequencing Prices
Costs are based on materials and time spent for preparation, which involves the type of study, multiplexing of
samples and depth of sequencing required per sample.
Additional Bioinformatics Analysis: Dependent upon time spent for analyses.
Contact
Dr. Keerthana (Kit) Krishnan: Keerthana@forsyth.org
Dr. Bruce Paster: bpaster@forsyth.org
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