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 . 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. 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. [email protected]. http://proteomics.forsyth.org/ 2 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: Cancer Markers Cardiovascular Cell Signaling Cellular Metabolism Immunology Genotyping Gene Expression Profiling Endocrinology Isotyping Matrix Metalloproteinases 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. 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 [email protected]. 3 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. [email protected]. 4 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. 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 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. 5 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. [email protected] Alexis Kokaras: 617-892-8567. [email protected] 6 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. [email protected] Steven Wilbert: 617-892-8552. [email protected] 7 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. [email protected]. 8 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 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: [email protected] Dr. Bruce Paster: [email protected] 9
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