Genomics Core Facility

Contact: Kristin Beaumont, PhD: Assistant Professor and Associate Director of Single Cell Genomic Technology Development (Kristin.beaumont@mssm.edu)

Online submission form link will be provided when project details are finalized. This submission form must be submitted prior to sample submission. Sample drop off is between 10AM and 3PM (2PM for CITESeq) in Icahn 13-76.

We offer a wide range of standard and custom single cell and spatial transcriptomic analysis approaches, designed around access to the following technology:

Sample Prep for Single Cell Analysis:

• Miltenyi GentleMACS Octodissociator for dissociation of tissues into single cell suspensions
• Levitas LeviCell sorting system (expected April 2021) for live cell/nuclei enrichment prior to single cell analysis

Single Cell/Nuclei Analysis:

• 10X Genomics Chromium system for high throughput 3’/5’ RNA Sequencing of thousands to tens of thousands of single cells or nuclei for subpopulation identification and expression analyses. We offer all immune profiling, CITESeq, ATACSeq, multiome RNA/ATACSeq and additional development based single cell assays using these instruments.

Input requirements: Debris-free suspension of at least 500,000 cells (>80% viability) for scRNASeq and scATACSeq and at least 1e6 cells (>80% viability) for CITESeq. Suspension should be at a concentration of 1e6 cells/mL in PBS + 0.04% BSA

• CellSee single cell platform for medium to high throughput cell selection and custom methods development applications. Input requirements vary by experiment – contact for details.
• MissionBio Tapestri platform for commercial and custom targeted amplicon DNA-sequencing at the single cell level. Input requirements vary by experiment – contact for details.
• Berkeley Lights Beacon  for isolation of up to 3500 single cells (or batches of cells) for single cell or low input real-time cellular biology in combination with various molecular pipelines for the characterization of selected single cells. Input requirements vary by experiment – contact for details.
• Fluidigm C1 for isolation of up to 96 single cells to then use for single cell sequencing purposes in combination with various molecular pipelines for the amplification of single cell RNA and DNA.

Spatial Transcriptomics

• 10x Genomics Visium: We are a Certified Service Provider for this assay, which is used for spatial transcriptomic profiling of tissues up to 6.5mm x 6.5mm.

Input requirements: Fresh frozen tissue banked in OCT block no larger than 6.5mm x 6.5mm

The Icahn Institute for Data Science and Genomic Technology and Department of Genetics and Genomic Sciences has made a substantial investment in high-throughput processing equipment for large BeadArray projects. We operate two TECAN Evo liquid handling robots, capable of processing 24 chips per run. Our Illumina HiScan microarray scanner is a top-of-the-line system with a robotic arm that allows for 24-hour microarray data acquisition. With the existing equipment and facilities, the genomics core can process up to 600 samples per week.

If you would like to submit samples for BeadArray analysis, please see sample submission page for location and pricing. Guidance for experimental design is also available.

For more information, see Illumina's site or view our specification sheet.

About Microarrays

DNA microarrays are a well-established technology for genome-wide characterization of gene transcription, single nucleotide variation, copy number variation, and epigenetic cytosine methylation. Our genomics core employs the most robust and accurate method on the market––the Illumina BeadArray platform.

Traditionally, microarrays were glass slides printed with short DNA strands. This method has serious drawbacks such as uneven spot morphology, signal bias due to spot position, and low design flexibility. BeadArrays are a unique approach to microarray technology, using glass slides with micrometer-sized holes to house oligonucleotide-coated beads. The BeadArrays are used as a standard array would be, with the exception of a molecular decoding step that is performed by the scanner.

The BeadArray platform can be used for genotyping, gene transcription quantification, and cytosine methylation.

Human genotyping studies are most often performed using the Infinium HD using OmniExpress (750k SNPs) or Omni2.5-8 arrays (2.4M SNPs). These chips are also capable of copy number variant estimation (CNV). Custom content chips for Genome Wide Association Studies (GWAS) are also available. For more information, please contact us with details of your project.

Gene expression analysis chips are available for humans, mice, and rats. All chips contain all known genes and many regulatory RNAs. As of March 2012, microRNA chips and custom gene expression services have been discontinued by Illumina.

Genome-wide methylation scanning in human samples (including stem cells and tumor cells) can be accomplished with the human methylation 450K array. This array covers CpG islands, sites known to be methylated in promoters, DNase hypersensitive sites and miRNA promoter regions.

Learn more about formalin fixed, paraffin embedded, samples that can be used for genotyping studies

Ion Torrent NGS

Contact: Ethan Ellis (ethan.ellis@mssm.edu)

We perform Ion Torrent sequencing on pre-existing and custom gene panels on the S5/XL system, including both AmpliSeq and non-AmpliSeq based panels. Our most frequently used panels are listed below. Please reach out to our team to learn more about these or other, more custom options.

Oncomine Comprehensive Assay v3:

A targeted next generation sequencing assay based on latest clinical oncology research for targeted solid tumor applications. This panel enables the detection of relevant SNVs, CNVs, gene fusions, and indels from 161 unique oncogenes. This panel is FFPE compatible and can accept DNA, RNA, or matched DNA/RNA samples with input as low as 10ng for each sample type.

Oncomine Myeloid:

A targeted, next generation sequencing assay that comprehensively covers all DNA variants and fusion transcripts known to be associated with myeloid malignancies. The panel includes 40 key target genes, 29 driver genes and a broad fusion panel including variants associated with AML, MDS, MPN, CML, CMML and JMML. Samples can be collected from blood or bone marrow, and the assay can be performed with inputs as low as 10ng RNA and 10ng DNA.

Cancer Hotspot Panel v2:

A targeted, next-generation sequencing assay that targets 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes focusing on genomic 'hot spot' regions that are frequently mutated in human cancer genes. This DNA-only panel can be run with as little as 10ng DNA and is FFPE compatible.

ReproSeq:

A comprehensive next generation sequencing solution for aneuploidy analysis. This panel enabled ploidy analysis of all 24 chromosomes (22 autosomes + X and Y chromosomes), and can detect copy number events as small as 17Mb. The panel is designed for processing a small number of cells from a trophectoderm biopsy, but any cell input or as little as 6pg of DNA can be utilized as input.

Contact person: Nancy Francoeur, PhD (nancy.francoeur@mssm.edu)

The online submission form must be submitted and project ID assigned prior to sample drop-off or shipment. Project ID should be printed on tubes/plates for submission. On-site sample drop off is between 10am-5pm Monday-Friday in Icahn 13-02.

We offer single molecule, real-time (SMRT) sequencing on the PacBio Sequel I and Sequel II systems. SMRT sequencing is characterized by long read lengths and high sequence accuracy, which can be used to sequence templates ranging from 250bp – 50kb. Common applications include de novo genome assembly, full-length transcriptome profiling and highly accurate amplicon sequencing. Please reach out to our team to learn more about the options listed below, or to inquire about other applications that may be under development.

Library Preparation Methods available:

Continuous long read (CLR) whole genome sequencing:

CLR WGS utilizes long insert library preparations, up to 30kb, from intact, high molecular weight (HMW) gDNA. The sequencing data generated from these libraries can be evaluated for the presence of a wide range of structural variants using a reference-based alignment approach, as well as be de novo assembled into highly contiguous reference genomes.

Input requirements: At least 5 μg HMW gDNA with average fragment sizes > 50kb.

HiFi whole genome sequencing:

HiFi WGS utilizes a slightly shorter library preparation, ranging from 10-18kb, that is tightly size selected prior to sequencing on either the Sequel I or Sequel II platforms. During sequencing, each molecule is sequenced repeatedly, and these multiple “passes” of the sequencing polymerase are collapsed in primary data processing to generate a single highly accurate (>99.9%) circular consensus sequence (CCS) per molecule. These data can be used to characterize SNPs and structural variants when compared to a reference genome.

Input requirements: At least 10 μg HMW gDNA with average fragment sizes >25kb

Low Input whole genome sequencing:

Although standard SMRT sequencing WGS library prep requires micrograms of gDNA input, libraries can also be prepared with as low as 300 ng gDNA when necessary to analyze very precious samples or single organism (i.e. insect) genomes.  Data from these runs are processed using the HiFi workflow described above and may be used for de novo genome assembly.

Input requirements: At least 300 ng HMW gDNA for genomes under 600 Mb; at least 400 ng of HMW gDNA for genomes up to 1 Gb, all with average fragment sizes >30 kb.

Ultra Low Input whole genome sequencing:

In circumstances where the amount of gDNA is extremely limited, the Ultra Low Input kit uses a whole-genome amplification (WGA) method to generate enough material for library preparation. Data from these runs are processed using the HiFi workflow described above and may be used for de novo genome assembly and variant analysis when compared to a reference genome.

Input requirements:  At least 5 ng HMW gDNA for genomes up to 500 Mb, with average fragment sizes >20kb

Isoform sequencing (IsoSeq):

IsoSeq libraries are constructed from cDNA generated by oligo dT priming of total RNA, in order to capture all polyadenylated transcripts. Sequencing data generation results in highly accurate HiFi reads per isoform, and initial primary analysis steps trim primer sequences and polyA tails, removes concatemers and performs de novo (reference-free) full-length isoform predictions. These data are typically used to generate novel reference transcriptomes, examine alternative splicing patterns or to characterize alternate promoter, exon and UTR usage under different experimental conditions.

Input requirements: At least 500ng total RNA, RIN > 7 or DV200 > 90%

Amplicon sequencing:

Amplicons generated from cDNA, DNA or bisulfite-treated DNA can be sequenced on the PacBio systems with high accuracy (>99.9%) and high contiguity, allowing for phasing of variants across the full amplicon length and providing haplotyping capabilities within a complex mixture. In the case of cDNA amplicons, targeted IsoSeq can be used to examine differential isoform usage or to resolve fusion transcripts at disease-relevant gene loci. Contact our team to discuss experimental design options and multiplexing strategies.

Input requirements: At least 500 ng nucleic acid template

HLA genotyping:

The Human leukocyte antigen (HLA) genes are some of the most polymorphic in the genome and play a key role in determining the quality of immune responses in the context of infectious disease, cancer and autoimmune disorders. HLA profiling allows investigators to assess the association of HLA alleles and resilience/susceptibility to disease. Using commercially available reagents and software from GenDx, our team offers profiling of class I and class II HLA genes. Depending on the profiling and throughput requested, up to 96 subject samples can be profiled per run.

Input requirements: 500-2000 ng gDNA from each individual to be profiled, dependent on loci screened

No-Amp targeted sequencing:

The No-Amp protocol utilizes the CRISPR-Cas9 system to enrich for large, complex genomic regions without the use of PCR amplification that can be subsequently sequenced on the Sequel systems. This application is typically used to resolve repeat expansions contributing to human disease, such as Fragile X Syndrome, ALS, Huntington’s disease and others. Contact our team for guide RNA design and multiplexing strategies.

Input requirements: 500-5000 ng HMW gDNA, depending on plex number

Pipeline FAQs:

HMW gDNA and RNA extraction services are also available and can be performed from cells, tissue, blood, OCT, buccal swabs, any vertebrate/invertebrate specimens and plants.

QC of DNA and RNA should be performed using a system that accurately quantifies DNA and RNA molecules with nucleic acid-specific kits, such as the Qubit Fluorometer or the Agilent systems, including Bioanalyzer, Tape Station, Fragment Analyzer or Femto Pulse. We do not recommend using a Nanodrop, as these systems measure all nucleotides in a solution and may vastly overestimate the quantity of material in a sample.

All sequencing services include all necessary QC and size selection of libraries.

Bioinformatics Services:

Demultiplexing of raw data and raw data delivery is always provided via secure FTP link as part of the sequencing cost. If requested, we are also able to perform all analysis pipelines available within SMRTLink, the Pacific Biosciences software associated with the Sequel systems, which are listed below. In these cases, an additional fee is included to cover compute costs.

Custom bioinformatic services are also available upon request, including large genome assemblies, HiFi genome assemblies, transcriptome annotation, targeted IsoSeq analysis and repeat expansion analysis, among others.

Circular consensus sequencing (CCS)

Also known as HiFi reads, the CCS algorithm removes any residual sequencing error by collapsing multiple sequencing reads over the same molecule, resulting in individual sequences with accuracy > 99.9%. Data delivered as FASTA/FASTQ CCS reads.

Hierarchical Genome Assembly (HGAP)

Typically used to assemble small, haploid genomes. Data delivered as FASTA/FASTQ assemblies.

Microbial Assembly (MA)

Similar to HGAP, but further tuned for particular features of microbial genomes, including circularizing assemblies and resolution of extrachromosomal features, such as plasmids. Data delivered as FASTA/FASTQ assemblies.

Base Modifications

Using the kinetics of base addition during SMRT-sequencing of native (not amplified) DNA templates, this analysis allows for the detection of DNA modifications and associated motifs, including 5-mC, 5-hmC, 5-fC, 5-caC, 4-mC, 6-mA, 8-oxoG and 8-oxoA.

IsoSeq

Initial CCS processing generates highly accurate reads per molecule sequenced. These reads are trimmed of primers and polyA tails, PCR artifacts (i.e. concatemers, heteroduplexes) are removed, and full-length isoforms are predicted de novo (i.e. reference-free).

Contact person: Aana Hahn (aana.hahn@mssm.edu)

We operate a full suite of Illumina NGS library preparation and sequencing pipelines, including DNA and RNA sequencing, as well as epigenetic profiling. Libraries are processed using the most cost-effective sequencing solution based on the project and batch sizes, including MiSeq, MiniSeq, NextSeq550, or NovaSeq instruments. We also accept pre-made libraries for sequencing. We provide troubleshooting assistance when needed to ensure successful completion of your project. We will also work with you for any custom requests that are not listed below.

Our Bioinformatics Team can deliver secondary data analysis or processing for an additional $75 per sample. Please see the NGS submission form for a listing of standard vs secondary deliverable options.

Please reach out to our team for any questions, and to receive a bespoke estimated cost table.

NGS submission form and policy can be found here: https://app.smartsheet.com/b/publish?EQBCT=4f134c08ae01452892689e5220890f5b

Library Preparation Methods Available:

RNA methods:

Illumina Stranded mRNA Prep:
RNA-seq library preparation method to specifically investigate mRNAs. Total RNA should be DNAase treated prior to submission. Kit is compatible with any species that has mRNA with polyadenylated 3’ ends. Not FFPE compatible.
Input requirements: total RNA with RIN >7; 25 to 1000ng input.

Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus:
RNA-seq library preparation that enables study of strand specific whole transcriptome library including protein coding mRNA as well as long non-coding RNA, while also preserving strand information. Small RNA will not be captured. The method is compatible with low quality, degraded and FFPE RNA samples. The kit specifically depletes abundant cytoplasmic RNA species (i.e. rRNA, mtRNA, globin RNA) from human, mouse and rat origin but may also be applicable to a variety of eukaryotic species (see link for species compatibility). Kit is FFPE compatible.
Input requirements: 1 to 1000ng total RNA input with RIN greater than or equal to 9, 10ng to 1000ng total RNA input with RIN greater than or equal to 2 or FFPE (DV200 greater than 55%), DNAse treated total RNA in nuclease-free water.

Takara SMART-Seq v4 Ultra Low Input:
RNA-seq library preparation used to specifically investigate mRNAs, from very low inputs of RNA (pg-ng). Polyadenylated RNA molecules are enriched via priming with oligo-dT. Total RNA should be DNAse treated prior to submission. Both cells and intact, extracted total RNA can be used as input into this protocol. Not FFPE compatible.
Input requirements: 1– 1,000 intact cells /sample suspended in up to 5 μl of Mg2+- and Ca2+-free cell culture-grade PBS or 100pg - 10ng DNAse treated total RNA with RIN >7 in nuclease-free water.

Agilent SureSelect XT RNA Direct:
Strand specific cDNA library preparation utilizing oligo-based capture to obtain mRNA information from degraded and FFPE-derived RNA samples. The capture library is based on the Agilent SureSelectXT Human All Exon v6 + UTRs.
Input requirments: 300ng of DNAse treated total RNA at 30ng/µl in nuclease-free water.

Takara SMARTer smRNA-Seq Kit for Illumina:
Library preparation method used to study small non-coding RNAs (including miRNAs) responsible for gene silencing and post-transcriptional regulation of gene expression.
Input: RIN>8, 1 ng–2 µg input of total RNA or enriched small RNA samples, DNAse treated, resuspended in nuclease-free water.

DNA methods:

Illumina DNA Prep:
Whole genome library preparation method, optimized for a wide range of input from 1-500 ng DNA for small genomes (e.g. microbial) and 100-500 ng for large genomes (e.g. human).
Input requirements: 300ng of intact gDNA at 20 ng/ µl in water, EB or low TE (<0.1mL EDTA)

Amplicon sequencing: Nextera XT and/or KapaHyper preparation methods:
We provide two methods for amplicon library prep: (1) Full length amplicon sequencing using direct ligation for small (< 500bp) using the KapaHyper preparation method; and (2) Larger amplicons (~10-20 kb) will require fragmentation prior to library prep using Nextera XT methods.
Input requirements: Small amplicons (<500 bp) 50 ng per amplicon; large amplicons (~10-20 kb), 10 ng, at a concentration of 200pg/ µl in water or EB; buffer must be EDTA-free.

Illumina DNA Prep with Enrichment:
DNA library preparation method for whole exome sequencing, in order to investigate all protein coding regions in a genome. Coding regions are captured by a panel of complimentary oligonucleotides for enrichment. This kit is panel agnostic. FFPE compatible.
Input requirements: 10ng to 1000ng genomic DNA, 50ng minimum for human samples, resuspended in water, EB, or low TE (<0.1mM EDTA).

Illumina TruSeq Methyl Capture EPIC Preparation:
DNA-based library method that identifies modified DNA bases, enriching the human methylome, specifically in CpG-rich regions both in and out of promoter sequences. Provides most agnostic solution to detect methylated bases, particularly when target is unknown.
Input requirements: 600ng intact gDNA at a concentration of at least 10ng/µl in water, EB or low TE (<0.1mM EDTA).

Takara DNA SMART ChIP-Seq Kit—Illumina® sequencing library preparation from low-input DNA templates:
The Takara DNA SMART ChIP-Seq protocol utilizes DNA recovered from the ChIP technique to produce amplified libraries with a set of unique indices suitable for sequencing on the Illumina sequencing platforms. dsDNA is denatured into ssDNA in order to add T-Tails to the template strands and replicate complementary strands with A-Tails. Both strands are tailed with oligonucleotide primers that are then template switched with a pair of forward and reverse primers. The complementary strands are then amplified through PCR.
Input Requirements: 0.1-10 ng bp ssDNA or dsDNA eluted from a ChIP protocol; Optimized for starting inputs of 200-400 bp.

User Prepared Libraries:
We are happy to accept and sequence user prepared libraries. Please submit >15nM library in >20 µl in water or EB (must be EDTA free) for direct QC and loading. Sequencing parameters and specifications can be discussed with our team.

Extractions:
Both DNA and RNA extractions are available through the facility. All Qiagen DNA or RNA extraction kits compatible with gDNA extraction from fresh or FFPE are supported, as well as custom requests. Qiagen QIAcube system allows for automated spin column based extractions to be performed in batches of 12. Prices available upon request.

QC Only Services:

Submission form and policy can be found here: https://app.smartsheet.com/b/form/4654d675a2d6456c92120a7c1a70e139

Qubit: Gold standard quantification of double-stranded DNA and RNA. Available in both broad range (DNA: 2-1000 ng; RNA: 20-1000 ng) and high sensitivity (DNA: 0.2-100 ng; RNA: 5-100 ng) configurations.

Agilent TapeStation or Agilent BioAnalyzer: Quantitation and visualization of DNA/RNA mass, integrity and size distribution. Provides RIN and DIN measurements of integrity critical for selecting appropriate preparation methods.

qPCR: Quantitation of specific templates. Available instrumentation provides throughput for 96-well and 384-well plates. User provides reagent and sample filled plate. Operator runs instrument and provides data values.

Shearing: Ultrasonic fragmentation of nucleic acids using the Covaris LE220 system.

Projects do not share a flowcell with other projects.