Center for Biomarker Research & Precision Medicine School of Pharmacy

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Our work leads to a variety of output that can help facilitate research of others.

Facilities

The Center for Biomarker Research and Precision Medicine (BPM) is housed in McGuire Hall and Annex on the university’s Medical College of Virginia Campus. The laboratory suite, covering over 2,000 square feet, includes a specialized genomics and epigenomics laboratory and a behavioral pharmacology laboratory. BPM investigators also have access to adjacent laboratory suite for epigenetic editing.

Genomics and Epigenomics Laboratory
Epigenetic Editing Laboratory
Behavioral Pharmacogenomics Laboratory
VCU Center for High Performance Computing
Scientist in lab using computer
Genomics and Epigenomics Laboratory

To minimize the risks of contamination and maximize throughout while keeping the quality of the data at the highest possible level, carefully optimized protocols and dedicated laboratory spaces are utilized for all projects

Sample registration and storage
All samples that are produced at, or arrive at, BPM are given a unique identifying barcode, which is integrated into our laboratory information management system. Using 1D and 2D barcodes this system allows for easy tracking of samples and applications with data stored in a centralized database that is backed-up remotely. For long term sample storage BPM has two Thermo Ultima II -80°C freezers on site in the McGuire facility, and maintains additional space for three Thermo Scientific High Prerformace -30 freezers in the Smith Building. Freezers are monitored 24/7 and investigators are automatically alerted of any unusual fluctuations in temperature.
Tissue handling and nucleic acid extraction
This laboratory is set up and approved for extraction of DNA and RNA from mammalian tissue, including extractions from human brain tissues, human whole blood and from dry archived blood spots.
DNA Fragmentation
This area is set up for fragmentation of genomic DNA for the downstream sequencing application. For sequencing of fragment libraries we typically shear the genomic DNA via ultrasonication using our Covaris™ S2 System (Covaris) with a Compact Chiller (VWR International). For large-scale projects we perform the same type of fragmentation in 96-well format using a Covaris E220 system, located at the VCU Genomics Core Laboratory. If larger fragment sizes are desired a Hydroshear (Genomic Solutions) is available.
Pre-PCR work
This laboratory is dedicated to pre-PCR procedures and houses our primary robotics platform, a Beckman Coulter Biomek® NXP robot, which is equipped with a span-8 head and gripper arm. This platform is frequently used for relatively straight forward pipetting activates such as dilutions and pooling of samples as well as for complex protocols such as library construction for next-generation sequencing.
Automated targeted amplification
This area is dedicated to targeted (bisulfite) amplicon preparation using the Juno platform (Fluidigm) for down-stream investigation via next-generation sequencing.
Nucleic acid quality control
This space is set up for rigorous quality and quantity control of all major steps of sample preparation and library construction. The instruments used for these procedures are mainly a BioAnalyzer 2100 (Agilent Technologies), a NanoDrop UV spectrometer (NanoDrop) and a Qubit 2.0 Fluorometer (Life Technologies) and digital gel imaging equipment (BioRad).
Next-generation sequencing
This laboratory houses our next-generation (ultra-high throughput) sequencing platforms. BPM owns a NextSeq 500 desktop sequencer (Illumina), which currently is the desktop sequencer with the highest output available as well as an MinION instrument (Oxford Nanopore technologies). An older next-generation sequencing platform, a SOLiD™ 5500xl Wildfire Genetic Analyzer (Life Technologies), is also available.
Pyrosequencing
This laboratory is equipped for high-throughput targeted pyrosequencing using a PyroMark Q96MD pyrosequencer (Qiagen).
Storage of sequencing data
The laboratory is hardwired to VCU’s Center for high performance computing facility, to which sequence data is auto exported and stored for further analysis. BPM we holds over 130 TB of dedicated storage space for (sequence) data.
Scientist injecting liquid into machine
Epigenetic Editing Laboratory

The epigenetic editing laboratory suite consists of two dedicated areas.

Microbiological work
In order to reduce the risk of cross-contamination, BPM houses a space reserved for microbiological work, such as routine cloning and host expansion, that is equipped with an incubator dedicated solely for bacterial culture.
Cell culture
The BPM investigators enjoys access to an, adjacently located, dedicated BSL-2+ cell culture laboratory in which BPM maintains a CO2 incubator for exclusive use. The culture room is equipped with a class II 1A biosafety cabinet (LabGard), inverted microscope, benchtop centrifuge, and local HEPA vacuum system. As such, the investigators are approved to use recombinant DNA products and produce lentiviral particles as part of research activities. The center also has access to a liquid nitrogen freezer for stocking of cell lines.
Scientist injecting liquid into trays
Behavioral Pharmacogenomics Laboratory

This laboratory is designed for studying alterations in behavior in response to treatment.

Behavioral pharmacology
This laboratory is specifically designed for behavioral testing of pharmacological response. It is equipped with computer-controlled, automated locomotor activity monitoring devices, pre-pulse inhibition devices and conditioning chambers.
Biomaterial extraction
This area is dedicated for biomaterial extraction and collection, primarily for studies of central nervous system drugs. For immediate fixation of tissue, the laboratory is equipped with a 10kW microwave tissue fixation system (Muromachi) and liquid N2.
Computer server racks
VCU Center for High Performance Computing

BPM is linked through a LAN and is hardwired to VCU’s high performance computing cluster (godel.vcu.edu).

High performance computing

The computing cluster consists of ~1200 Opteron 64 bit and Intel 64 bit cores, each with at least 3 GB RAM/core, and 4.2TB of total RAM. The servers are connected by QDR Infiniband connections. Connected to this cluster BPM has 130 TB storage space that is exclusively dedicated for the center’s use.

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Protocols

Methyl-binding domain (MBD) enrichment
DNA extraction from dry blood spots
Methyl-binding domain (MBD) enrichment

We use a carefully optimized protocol for methyl-binding domain (MBD) enrichment in combination with high output next-generation sequencing to assess CpG methylation.

The approach is evaluated and described in

Chan RF, Shabalin AA, Xie LY, Adkins DE, Zhao M, Turecki G, Clark SL, Aberg KA, van den Oord EJCG. Enrichment methods provide a feasible approach to comprehensive and adequately powered investigations of the brain methylome. Nucleic Acids Res. 2017 Jun 20;45(11):e97.

 

Aberg KA, Chan RF, Shabalin AA, Zhao M, Turecki G, Staunstrup NH, Starnawska A, Mors O, Xie LY, van den Oord EJ. A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA. Epigenetics. 2017 Sep;12(9):743-750.

DNA extraction from dry blood spots

The following section includes modified protocols for extraction of DNA We have found the DNA extracted with these protocols to be of high quality and suitable for next-generation sequencing based approaches.

DNA extraction of a complete blood spot or left over scrap material

We have recently evaluated the ability to use DNA extracted from dry blood spots for methylome-wide investigations. We have published a modified version of a DNA protocol that allowed for extraction of the DNA from a complete blood spot in one reaction (Aberg et al. Epigenetics 2013 May;8(5):542-7.).

Protocol for DNA extraction of a complete blood spot in a single reaction

We have further modified our blood spot extraction protocol to also allow for efficient extraction of DNA from scrap material, i.e., the part of the blood spot that is left when punches, typically used for standard DNA extraction protocols, have been removed.

Protocol for Genomic DNA Isolation from Dried Blood Spots‌

Software

RaMWAS
lga972
ShiftR
RaMWAS

A Bioconductor package for Fast Methylome-Wide Association Study Pipeline for Enrichment Platforms

A complete toolset for methylome-wide association studies (MWAS). It is specifically designed for data from enrichment based methylation assays, but can be applied to other data as well. The analysis pipeline includes seven steps: (1) scanning aligned reads from BAM files, (2) calculation of quality control measures, (3) creation of methylation score (coverage) matrix, (4) principal component analysis for capturing batch effects and detection of outliers, (5) association analysis with respect to phenotypes of interest while correcting for top PCs and known covariates, (6) annotation of significant findings, and (7) multi-marker analysis (methylation risk score) using elastic net. Additionally, RaMWAS include tools for joint analysis of methlyation and genotype data.

Shabalin A, Hattab M, Clark S, Chan R, Kumar G, Aberg K, van den Oord E (2018). “RaMWAS: fast methylome-wide association study pipeline for enrichment platforms.” Bioinformatics.
LGA972

A cross-platform application for optimizing LD studies

Because of the assays costs and large sample sizes that are required to discover effects while controlling false discoveries, large scale genetic association studies can be very expensive. Two-stage designs can be used to design these studies in the most cost-effective way. In two stage designs all the markers are assayed and tested in a first stage. Only the promising markers are subsequently assayed in the second stage using additional samples. Compared to single-stage studies, optimized multistage designs can achieve the same goals in terms of true and false discoveries with a 50-70% saving in the amount of genotyping. Furthermore, rather than using arbitrary rules (e.g. P-values smaller than 0.05 suggest a replication), use of multistage designs can provide statistically motivated decision rules for declaring significance.

lga972 is a cross-platform application with a graphical interface that uses a genetic algorithm for determining the design features of 2-stage genetic association studies that minimize the genotyping burden. The user can choose among a variety of case-control and family based tests where outcome may be scored as present versus absent or is a continuous variable. The text-based output can easily be exported to other programs such as word-processors and spreadsheets.

Lga972 is described in

Robles, J & Van den Oord, EJCG (2004). lga972: A cross-platform application for optimizing LD studies via the genetic algorithm. Bioinformatics, 20, 3244-3245. Related papers:

ShiftR

Fast Enrichment Analysis via Circular Permutations

After testing many markers for association with an outcome, researcher often examine whether their top findings are enriched for specific (epi-)genomic features, pathways, or findings from other association studies. Standard statistical tools (e.g., Fisher’s exact test) may not be applicable as they would produce too optimistic P values due to the dependency of observations or use of multiple thresholds to select the top results.

To address these challenges we created shiftR, an R package for enrichment testing through fast circular permutations. shiftR is free, cross-platform, open source, and applicable for datasets of any type. A very fast algorithm based on bitwise operations is used to enable calculation of permutation P values with high dimensional data sets.

A vignette showing how to use shiftR can be found here

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