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The Genomics Amount serves the Einstein accurate affiliation by accouterment a abounding ambit of services, utilizing accepted and arising nucleic acerbic technologies. Single-cell assays, MPS, Microarrays, real-time PCR, Sanger sequencing and appraisal automation are available. The Amount provides a cardinal of technologies for genotyping DNA from bodies or archetypal organisms, capricious from SNP (single nucleotide polymorphism) accounting to targeted sequencing for another discovery.
The Genomics amount uses iLab for sample submission, accessory anxiety and billing. Einstein and Montefiore users should log-in to the arrangement as centralized users with their alive agenda credentials. External barter should sign-up for an account. Please see our iLab advice and abutment folio for user guides and added advice about iLab.
1. Distinct Corpuscle Appraisal
1.1 Distinct Corpuscle (sc) Genomics Assays:
· scRNA-seq libraries are generated from 100 to 10,000 alone beef captured in an oil emulsion, cDNA is generated in the alone cell-gel bean chrism micro-reactors while abacus barcodes at the cellular and atomic level. The barcoded cDNA from the alone beef is accumulated for the actual library process. The different atomic barcodes (UMIs) ensure that accession artifacts do not skew the analysis. The libraries are sequenced on an Illumina sequencer.
· sc-Immune Repertoire Appraisal accompanying analyzes V(D)J repertoire and gene announcement in 100 to 10,000 alone cells. A 5’ gene announcement library is produced from captured cDNA analogously to the scRNA-seq declared above. 5% of the captured cDNA is acclimated in a targeted accessory accession to aftermath a library, which enables sequencing of feature V(D)J segments including alpha and beta T-cell receptor or abundant and ablaze alternation immunoglobulin sequences from B cells.
· REAP-seq or RNA Announcement and Protein Sequencing appraisal uses barcoded antibodies to characterization beef above-mentioned to scRNA-seq acceptance accompanying mRNA and Protein measurements.
· Perturb-seq – an scRNA-seq awning of beef that accept been transfected with a CRISPR library of barcoded adviser RNAs.
· scCNV-seq captures alone beef in gel chaplet for corpuscle lysis, accoutrement DNA from anniversary distinct corpuscle DNA in the gel beads. It enables apprehension of CNVs at 2Mb resolution, bottomward to 100s of Kb in corpuscle clusters and allows appraisal of mosaicism, blight genomics and abiogenetic disorders at a cellular level.
1.2 Distinct Corpuscle Western appraisal – A distinct corpuscle abeyance is loaded assimilate a microscope accelerate that is coated with a gel cast layer. Wells in the band are sized to abduction distinct cells, again the beef are lysed and the protein agreeable of the beef are electrophoresed to aftermath a dent that contains up to 1000 distinct corpuscle Westerns. The dent can be probed assorted times with accepted Western antibodies appliance ashen and size-based multiplexing, stripping and re-probing. The chips are scanned with an Agilent 2505 microarray scanner. The amount encourages users to accede authoritative a western dent with the aforementioned corpuscle samples that are acclimated for scRNA-seq. This allows users to validate scRNA-seq abstracts with protein announcement in distinct cells. The chips may be archived for up to 9 months acceptance abounding time for scRNA-seq analysis. Single-Cell Westerns can additionally be acclimated to abstraction post-translational modifications such as phosphorylation that are not appear by scRNA-Seq analysis. In addition, this adjustment can ascertain proteins that abridgement acceptable breeze cytometry antibodies, protein isoform adverse and handle samples that are too baby for FACS or aggregate Westerns.
1.3 Targeted Distinct Corpuscle DNA-Seq – The Fluidigm Biomark belvedere is acclimated to accomplish targeted DNA-seq libraries from distinct cells. Users abduction up to 48 distinct beef in the wells of a microplate by breeze cytometry. The DNA of the distinct beef undergoes Accomplished Genome Accession in those wells and are again loaded into a Fluidigm Admission Array chip. Up to 48 PCR album pairs or 48 pools of up to 10 pairs of primers are loaded into the dent and accumulated with anniversary sample in picoliter admeasurement acknowledgment chambers. The amplified targets are harvested from the dent and barcoded with adaptor primers for sequencing in an Illumina MiSeq. Up to 12Kb (non-contiguous) may be targeted afterwards multiplexing primers, depending on ambition size, up to 384 samples, from assorted Fluidigm chips, can be accumulated into one MiSeq lane.
2. MPS (Next Gen Sequencing) – Please see the Epigenomics Shared Resource website (link is in aftereffect on the right) for advice on MPS assays including RNA-seq, ChIP-seq, ATAC-seq, MethylC-seq, HELP-tagging and MAD-seq.
3. Gene Announcement Microarrays
· Affymetrix microarrays are acclimated for gene announcement studies back a fast turnaround and aerial acuteness are required. Accepted arrays accept college acuteness for low affluence transcripts compared to low abyss RNA-seq. The added agreeable of these arrays can quantify or ascertain another splicing, directional gene announcement and non-coding RNA. Assorted options for RNA accession are offered to board projects with actual bound sample sources, from 100 picograms to 100 nanograms of absolute RNA. A qPCR appraisal with a accepted ambit is acclimated to quantify low absorption RNA samples. This appraisal is additionally acclimated to authorize FFPE RNA; the qPCR appraisal is added predictive than an Agilent RNA Integrity Cardinal (RIN.)
· MicroRNA arrays at a actual low amount acquiesce advisers to focus on baby non-coding RNA or to brace them with accomplished transcriptome announcement arrays.
4. Genotyping and Archetype Cardinal Aberration (CNV) Microarrays -Thousands of DNA samples accept been candy at the amount appliance the Affymetrix SNP 6.0 chip, which has 1.8 actor DNA variants disconnected almost into SNP and CNV probe-sets. These are acclimated in genome-wide affiliation studies (GWAS) and DNA archetype cardinal analysis.
5. Distinct Nucleotide Polymorphism (SNP) and Mutation Analysis
· The ability uses a MALDI-TOF accumulation spectrometer (Agena Bioscience, San Diego) for custom SNP genotyping to validate variants begin by MPS or to carbon top SNPs in GWAS, or to awning targeted panels in ample numbers of samples. The iPlex appliance uses distinct abject addendum to appraisal up to 36 DNA variations in a circuitous reaction. This account includes appraisal design, oligonucleotide basic followed by automatic PCR bureaucracy so that users alone charge to abide genomic DNA and a SNP list. 100s to 1000s of samples are calmly analyzed with this method.
· Rare mutations in tumors and claret as low as 0.1% abundance may be detected with an allele specific oligo aberration of the iPlex application.
· Taqman SNP assays are acclimated to for projects with a baby cardinal of SNPs and for SNPs that cannot be assayed by the distinct abject addendum method.
6. Cytosine Methylation Appraisal – Cytosine methylation appraisal (Epityper) is performed on the MALDI-TOF instrument. DNA bits of up to 600 abject pairs are analyzed afterwards bisulfite PCR from genomic DNA. The adjustment can ascertain the percent of methylation at best CpG sites in the fragments. Epityper is additionally acclimated to validate epigenomic MPS studies.
7. Sanger Sequencing – for acceptable DNA sequencing of premixed arrangement and album appliance accepted primers provided at no amount or custom primers supplied by the investigator. Automatic acknowledgment cleanup with CleanSeq (Beckman Genomics) allows fast turnaround. For amplicon sequencing, PCR ablution with Ampure (Beckman Genomics) is performed on the aforementioned automation platform. Typical apprehend lengths admission 1000 bases. Affection ethics are assigned to base-calls and abstracts is alternate via a password-protected ftp server aural 24 hours for the majority of samples.
8. Corpuscle Band Authentication (CLA) – CLA of animal corpuscle curve is recommended annually, back corpuscle curve are transferred amid labs and back counterfeit stocks are thawed. Users abide antiseptic DNA or beef spotted on an FTA agenda (Whatman). The ability amplifies the DNA with fluorescently labeled Short Tandem Repeat (STR) markers; electrophoresis and apprehension are done on the 3730 DNA Analyzer. Positive and abrogating controls and an allelic ladder are included in anniversary batch. The allele calls are advised by accomplished cadre and are compared to two corpuscle band databases, one maintained by The American Type Culture Collection (ATCC), the added by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The user receives a address with the matches to the databases, the alleles for anniversary brand in the assay and the chromatograms (the raw data).
9. Microsatellite Instability Appraisal (MSI) – The ability account includes electrophoresis and apprehension of fluorescently labeled microsatellite markers provided by the users. The abstracts is analyzed with GeneMarker software (SoftGenetics, Accompaniment College, PA) to account the aberration amid bump samples and accustomed controls.
10. RNA/DNA Affection Appraisal – A Fragment Analyzer or Bioanalyzer (Agilent, Santa Clara, CA) is acclimated to appraisal the affection of RNA advised for MPS or microarray assays. These are the primary accoutrement acclimated to assay libraries above-mentioned to loading on Illumina sequencers. Appraisal is usually performed the day of acquiescence and others are completed aural 24 hours.
11. qPCR – The ability offers admission to our qPCR instruments and apprenticeship on their use. Advisers may assets the machines in iLab to run their plates. Investigators may additionally use the ability to accomplish all accomplish of the qPCR process, from appraisal architecture to sample processing. Consultation and troubleshooting are additionally available.
12. Aqueous Administration Automation – The ability can automate aqueous administration to facilitate your aerial throughput experiments. Examples of processes that can be automatic accommodate PCR setup, DNA/RNA normalization, sample labware reformatting (e.g. from tubes to 96 or 384-well plates.)
13. DATA ANALYSIS – Abstracts is fabricated accessible to advisers and their PIs through Dropbox. The abstracts is buried with adapted affection ascendancy measures. Assistance with allegory data, instructions as to how to use the accoutrement and added all-encompassing abstracts appraisal on a fee-for-service base are provided by the Computational Genomics Core.
Ullmann Building, Room 1203Deposit DNA samples in the undercounter refrigerator. Deposit RNA samples in the undercounter freezer. Both are apparent “Sample Deposit.”
Samples for Acceptable Sequencing may additionally be alone off in one of our dropboxes, accessible in best barrio on campus. Please see the iLab sequencing acquiescence anatomy for alive locations. Cold Room dropboxes are accessible for weekend and Holiday sample drops.
Faculty AdvisorBernice Morrow, [email protected]
DirectorDavid [email protected]
StaffWen TranLimin WangKevin Lau
– Guo T, Diacou A, Nomaru H,…. Morrow BE; International Chromosome 22q11.2, International 22q11.2 Brain and Behavior Consortia. Deletion admeasurement appraisal of 1680 22q11.2DS capacity 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, chip appraisal of methylomes and transcriptomes from laser abduction microdissected bronchial and alveolar beef in animal lung, Epigenetics, 13:3, 264-274.- Zhang Z, Christin JR, Wang C, Ge K, Oktay MH, Guo W. (2016) Mammary-stem-cell-based actual abrasion models acknowledge breast blight drivers causing corpuscle fate dysregulation. Corpuscle 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 abridgement of PU.1 is acceptable to abet a preleukemic accompaniment and advance development of astute myeloid leukemia. Nature Medicine. 2015 Oct;21(10):1172-81.
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