Mission
The Genomics Core serves the Einstein scientific community by providing a broad range of services, utilizing current and emerging nucleic acid technologies. Single-cell assays, MPS, Microarrays, real-time PCR, Sanger sequencing and assay automation are available. The Core provides a number of technologies for genotyping DNA from humans or model organisms, varying from SNP (single nucleotide polymorphism) typing to targeted sequencing for variant discovery.
Access Services
The Genomics core uses iLab for sample submission, device reservations and billing. Einstein and Montefiore users should log-in to the system as internal users with their active directory credentials. External customers should sign-up for an account. Please see our iLab help and support page for user guides and more information about iLab.
Services
1. Single Cell Analysis
1.1 Single Cell (sc) Genomics Assays:
· scRNA-seq libraries are generated from 100 to
10,000 individual cells captured in an oil emulsion, cDNA is generated in the
individual cell-gel bead emulsion micro-reactors while adding barcodes at the
cellular and molecular level. The
barcoded cDNA from the individual cells is combined for the remaining library
process. The unique molecular barcodes
(UMIs) ensure that amplification artifacts do not skew the analysis. The libraries are sequenced on an Illumina
sequencer.
· sc-Immune Repertoire Analysis simultaneously
analyzes V(D)J repertoire and gene expression in 100 to 10,000 individual
cells. A 5’ gene expression library is
produced from captured cDNA similarly to the scRNA-seq described above. 5% of the captured cDNA is used in a targeted
enrichment amplification to produce a library, which enables sequencing of
full-length V(D)J segments including alpha and beta T-cell receptor or heavy
and light chain immunoglobulin sequences from B cells.
· REAP-seq or RNA Expression and Protein
Sequencing assay uses barcoded antibodies to label cells prior to scRNA-seq
allowing simultaneous mRNA and Protein measurements.
· Perturb-seq - an scRNA-seq screen of cells that
have been transfected with a CRISPR library of barcoded guide RNAs.
· scCNV-seq captures individual cells in gel beads
for cell lysis, trapping DNA from each single cell DNA in the gel beads. It enables detection of CNVs at 2Mb
resolution, down to 100s of Kb in cell clusters and allows analysis of
mosaicism, cancer genomics and genetic disorders at a cellular level.
1.2 Single Cell Western analysis – A single cell suspension is
loaded onto a microscope slide that is coated with a gel matrix layer. Wells in the layer are sized to capture
single cells, then the cells are lysed and the protein content of the cells are
electrophoresed to produce a chip that contains up to 1000 single cell
Westerns. The chip can be probed
multiple times with standard Western antibodies using spectral and size-based
multiplexing, stripping and re-probing.
The chips are scanned with an Agilent 2505 microarray scanner. The core encourages users to consider making
a western chip with the same cell samples that are used for scRNA-seq. This allows users to validate scRNA-seq data
with protein expression in single cells.
The chips may be archived for up to 9 months allowing ample time for
scRNA-seq analysis. Single-Cell Westerns
can also be used to study post-translational modifications such as
phosphorylation that are not revealed by scRNA-Seq analysis. In addition, this method can detect proteins
that lack good flow cytometry antibodies, protein isoform heterogeneity and
handle samples that are too small for FACS or bulk Westerns.
1.3 Targeted Single Cell DNA-Seq - The Fluidigm Biomark platform is
used to generate targeted DNA-seq libraries from single cells. Users capture up to 48 single cells in the
wells of a microplate by flow cytometry.
The DNA of the single cells undergoes Whole Genome Amplification in
those wells and are then loaded into a Fluidigm Access Array chip. Up to 48 PCR primer pairs or 48 pools of up
to 10 pairs of primers are loaded into the chip and combined with each sample
in picoliter size reaction chambers. The
amplified targets are harvested from the chip and barcoded with adaptor primers
for sequencing in an Illumina MiSeq. Up
to 12Kb (non-contiguous) may be targeted without multiplexing primers, depending
on target size, up to 384 samples, from multiple Fluidigm chips, can be combined
into one MiSeq lane.
2. MPS (Next Gen Sequencing) – Please see the Epigenomics Shared Resource
website (link is in sidebar on the right) for information on MPS assays
including RNA-seq, ChIP-seq, ATAC-seq, MethylC-seq, HELP-tagging and MAD-seq.
3. Gene Expression Microarrays
· Affymetrix microarrays are used for gene
expression studies when a fast turnaround and high sensitivity are
required. Current arrays have higher
sensitivity for low abundance transcripts compared to low depth RNA-seq. The
increased content of these arrays can quantify or detect alternative splicing,
directional gene expression and non-coding RNA. Multiple options for RNA
amplification are offered to accommodate projects with very limited sample
sources, from 100 picograms to 100 nanograms of total RNA. A qPCR assay with a standard curve is used to
quantify low concentration RNA samples.
This assay is also used to qualify FFPE RNA; the qPCR assay is more
predictive than an Agilent RNA Integrity Number (RIN.)
· MicroRNA arrays at a very low cost allow
researchers to focus on small non-coding RNA or to pair them with whole
transcriptome expression arrays.
4. Genotyping and Copy Number Variation (CNV) Microarrays -Thousands
of DNA samples have been processed at the core using the Affymetrix SNP 6.0
chip, which has 1.8 million DNA variants divided roughly into SNP and CNV
probe-sets. These are used in genome-wide association studies (GWAS) and DNA
copy number analysis.
5. Single Nucleotide Polymorphism (SNP) and Mutation Analysis
· The facility uses a MALDI-TOF mass spectrometer
(Agena Bioscience, San Diego) for custom SNP genotyping to validate variants
found by MPS or to replicate top SNPs in GWAS, or to screen targeted panels in
large numbers of samples. The iPlex application uses single base extension to
analyze up to 36 DNA variations in a multiplex reaction. This service includes assay design,
oligonucleotide prep followed by automated PCR setup so that users only need to
submit genomic DNA and a SNP list. 100s
to 1000s of samples are easily analyzed with this method.
· Rare mutations in tumors and plasma as low as
0.1% frequency may be detected with an allele specific oligo variation of the
iPlex application.
· Taqman SNP assays are used to for projects with
a small number of SNPs and for SNPs that cannot be assayed by the single base
extension method.
6. Cytosine Methylation Analysis - Cytosine methylation analysis
(Epityper) is performed on the MALDI-TOF instrument. DNA fragments of up to 600
base pairs are analyzed after bisulfite PCR from genomic DNA. The method can detect the percent of
methylation at most CpG sites in the fragments. Epityper is also used to
validate epigenomic MPS studies.
7. Sanger Sequencing - for traditional DNA sequencing of premixed
template and primer using standard primers provided at no cost or custom
primers supplied by the investigator.
Automated reaction cleanup with CleanSeq (Beckman Genomics) allows fast
turnaround. For amplicon sequencing, PCR
purification with Ampure (Beckman Genomics) is performed on the same automation
platform. Typical read lengths approach
1000 bases. Quality values are assigned
to base-calls and data is returned via a password-protected ftp server within
24 hours for the majority of samples.
8. Cell Line Authentication (CLA) - CLA of human cell lines is
recommended annually, when cell lines are transferred between labs and when
unverified stocks are thawed. Users submit
purified DNA or cells spotted on an FTA card (Whatman). The facility amplifies the DNA with
fluorescently labeled Short Tandem Repeat (STR) markers; electrophoresis and
detection are done on the 3730 DNA Analyzer.
Positive and negative controls and an allelic ladder are included in
each batch. The allele calls are
reviewed by experienced personnel and are compared to two cell line databases,
one maintained by The American Type Culture Collection (ATCC), the other by the
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The user receives a report with the matches
to the databases, the alleles for each marker in the test and the
chromatograms (the raw data).
9. Microsatellite Instability Analysis (MSI) - The facility service
includes electrophoresis and detection of fluorescently labeled microsatellite
markers provided by the users. The data
is analyzed with GeneMarker software (SoftGenetics, State College, PA) to score
the difference between tumor samples and normal controls.
10. RNA/DNA Quality Analysis – A Fragment Analyzer or Bioanalyzer (Agilent,
Santa Clara, CA) is used to assay the quality of RNA intended for MPS or
microarray assays. These are the primary
tools used to check libraries prior to loading on Illumina sequencers. Analysis
is usually performed the day of submission and others are completed within 24
hours.
11. qPCR - The facility offers access to our qPCR instruments and
instruction on their use. Researchers
may reserve the machines in iLab to run their plates. Investigators may also use the facility to perform all steps of
the qPCR process, from assay design to sample processing. Consultation and troubleshooting are also
available.
12. Liquid Handling Automation - The facility can automate liquid
handling to facilitate your high throughput experiments. Examples of processes that can be automated
include PCR setup, DNA/RNA normalization, sample labware reformatting (e.g.
from tubes to 96 or 384-well plates.)
13. DATA ANALYSIS – Data is made available to researchers and their PIs through Dropbox. The data is screened with appropriate quality control measures. Assistance with analyzing data, instructions as to how to use the tools and more extensive data analysis on a fee-for-service basis are provided by the Computational Genomics Core.
Location
Ullmann Building, Room 1203
Deposit DNA samples in the undercounter refrigerator. Deposit RNA samples in the undercounter freezer. Both are marked “Sample Deposit.”
Samples for Traditional Sequencing may also be dropped off in one of our dropboxes, available in most buildings on campus. Please see the iLab sequencing submission form for active locations. Cold Room dropboxes are available for weekend and Holiday sample drops.
Personnel
Faculty Advisor
Bernice Morrow, Ph.D.
718.678.1121
morrow@einsteinmed.org
Director
David Reynolds
929.246.6735
david.reynolds@einsteinmed.org
Staff
Wen Tran
Limin Wang
Kevin Lau
Phone: 929.246.6735
Selected Publications
- Guo T, Diacou A, Nomaru H,…. Morrow BE; International Chromosome 22q11.2, International 22q11.2 Brain and Behavior Consortia. Deletion size analysis of 1680 22q11.2DS subjects identifies a new recombination hotspot on chromosome 22q11.2. Hum Mol Genet. 2018 Apr 1;27(7):1150-1163.
- Dong X, Shi M, Lee M, Toro R, Gravina S, Han W, Yasuda S, Wang T, Zhang Z, Vijg J, Suh Y & Spivack SD (2018) Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung, Epigenetics, 13:3, 264-274.
- Zhang Z, Christin JR, Wang C, Ge K, Oktay MH, Guo W. (2016) Mammary-stem-cell-based somatic mouse models reveal breast cancer drivers causing cell fate dysregulation. Cell Reports 16: 3146-3156.
- Will B, Vogler TO, Narayanagari S, Bartholdy B, Todorova TI, Chen J, Yi Yiting, Mayer J, daSilva Ferreira M, Barreyro L, Carvajal L, Roth M, van Oers J, Schaetzlein S, McMahon C, Edelmann W, Verma A, Steidl U. Minimal reduction of PU.1 is sufficient to induce a preleukemic state and promote development of acute myeloid leukemia. Nature Medicine. 2015 Oct;21(10):1172-81.