Epigenomics Core @ WCMC

Epigenomics Core @ WCMC
Service Request Workflow
GETTING STARTED:
What you need to do
Create an account in the web-based
Agilent Crosslab/iLab Service Request LIMS
by following the instructions under
GETTING STARTED
Set up payment information in iLab
Internal customers
(Weill Cornell Medicine) require
Fund Numbers
for work to begin. Please follow the instructions in the
WCMC iLab Instructions PDF
to add fund numbers to your Agilent Crosslab/iLab account.
External academic customers
- please ensure that you include details for your PI and financial administrator and that your financial manager enters a
correct billing address
in their Agilent Crosslab/iLab account. Please use this
W-9 Tax form
if required, towards setting up payment.
Request a service in
Agilent Crosslab/iLab Service Request LIMS
Request Services
Page by selecting from our options menu.
Click the
Initiate Request
button to the right of the service and follow the instructions for the service to submit an online request.
Notes:
Make sure to fill in the appropriate sample/library submission table in Crosslab depending on the service that you are requesting. (NOTE: Please
de-identify any patient samples
before submitting them to us.)
Quality Control is included
in the sequencing requests.
Please wait for the Epigenomics Core to Approve the request and provide your lab with a Price Quote. This may take up to 48 hrs.
Once approved, your quote can be downloaded as a PDF from within your Agilent Crosslab/iLab request.
Requests without financial approval will not be honored:
Internal customers must have a valid
Fund Code
and
Approve
the Price Quote.
External Academic Customers must
update the payment information in Agilent Crosslab/iLab
once they have obtained a
Purchase Order Number
for the quoted price.
After financial approval in
Agilent Crosslab/iLab
print out the sample submission form
, make sure you enter the
Agilent Crosslab/iLab
service ID in the appropriate field.
Samples will not be processed without an accompanying
Agilent Crosslab/iLab
request.
Label the individual tubes clearly with the
sample number you have indicate in the sample submission form AND Agilent Crosslab/iLab service IDs
Samples requiring dry ice (RNA, ChIPd) or ice temperatures (DNA) must be in an
appropriate insulated container
with the samples protected inside the shipping container. [Please reference the service you would like to use for specific details of sample submission.]
Ship samples
with
sample submission form to:
Epigenomics Core Facility
Weill Cornell Medicine
413 East 69th Street, Room A-427
New York, NY 10065
OR
Bring samples
with
sample submission form to:
Epigenomics Core Facility at Weill Cornell Medical College,
1300 York Avenue, A-427,
New York, NY 10065
If you are dropping off your samples please do so between 9:30am and 4:30pm on business days
Invoice will be generated after completion of the project; internal customers will be billed through SAP, External Academic customers please refer to
payment instructions
on our website.
CORE OPERATING PROCEDURES:
Quality control is performed on all samples before sequencing using Qubit Fluorometer, Agarose Gels and/or Bioanalyzer Chip depending on the type of assay. Please click through our
(Epi)Genetic Services
menu for more information on the sample requirements and quality control for the assay you are interested in.
Samples that pass our quality checks are processed for library preparation and/or sequencing as requested. Please click through our
(Epi)Genetic Services
menu to find details about Library Preparation and Sequencing.
After your samples are sequenced, we summarize and demultiplex the base calls as required into raw data files with quality information and then use post-processing pipelines optimized to specific sequencing assays (upon request) to generate alignments, methylation calls, summary statistics etc. Please review the
Data Processing
section in the assay of your choice in on our
(Epi)Genetic Services
menu for more information.
Sequencing data processed at the core is distributed via
Pubshare
and stored for 2 years. Please note that Pubshare and iLab are two separate systems requiring separate accounts and login information. For more information on data retrieval, please visit the
Data Analysis and Retrieval section of our FAQ
FINISHING UP:
For customers paying from the Weill Cornell Medical College:
Fund Number
valid for the entire length of the service in
Agilent Crosslab/iLab
is required.
To manage Fund Numbers on your
Agilent Crosslab/iLab
page
please follow the instructions in the
WCMC iLab Instructions PDF
Payment will be processed automatically through WBG/SAP after an Invoice is issued (at the end of the calendar month following the completion of the service).
If your Fund Number is no longer billable for any reason, please contact us at
epigenomicscore@med.cornell.edu
For customers from Cornell University, Ithaca:
Please pay by
Fund Transfer from CU to WCMC
using your central institutional account, and include a copy of the invoice. Reach out to your accounting personnel for directions and clarification on how to process internal transfers between our campuses.
Once you have posted the transfer, please email us at
all2041@med.cornell.edu
in order to ensure credit to your iLab account.
For External Academic customers:
External Academic customers must pay via
check or wire transfer ONLY, we do not accept credit cards.
After completion of the service an invoice referencing the provided PO number will be generated.
The
Invoice will be emailed
through iLab to the Financial Manager and/or PI - who is responsible for delivering it to the Institution's Accounts Payable Department.
Your Institution’s Accounts Payable Department should mail a check payable to
Weill Cornell Medical College
along with a copy of your invoice to:
Epigenomics Core Facility
Weill Cornell Medical College, Box 288
1300 York Avenue
New York, NY 10065
If you are paying via a wire transfer please send an email to
all2041@med.cornell.edu
to arrange the proper transfer.
If you have any questions about your invoice, please email
epigenomicscore@med.cornell.edu
All inquires regarding billing should be addressed to
all2041@med.cornell.edu
Forms
W-9 Tax form for billing (External Academic customers only)
TIP:
click on the orange buttons to expand (or close) each section in the workflow.
(Epi)Genetic Profiling Services
Overview
Please Note:
We have changed our sample drop off protocols and times - click on and read the following PDFs for details:
Bulk Sequencing Sample Submission
Single Cell Sequencing Sample Submission
RNASeq, DNASeq and sequencing are performed by the Weill Cornell Medicine Genomics Resources Core Facility (GRCF).
If you require these services
- please submit your samples directly to the
GRCF
. Please contact
Dr. Jenny Xiang
if you have any questions about these services.
The core accepts samples that require library preparation and sequencing.
There will be a quality control step prior to agreement of service. Please refer to specific services using the menu on the left for detailed quality control descriptions.
The price for library preparation is per library
, and includes sample quality control, library preparation and library validation.
The price for sequencing varies based on sample volume and number of reads required per sample
, and includes de-multiplexing (if required), post-processing (if available and requested) and two years data storage. For methylation sequencing services, methylation calls are also provided with alignments as part of the post-processing.
Sequencing is performed on
NovaSeq 6000
or
MiSeq
Illumina
instruments.
A Bioinformatics Fee (10% of the sequencing price for Internal Clients and 20% for External Clients) will be added.
Libraries made by the core, routinely yield clusters between 750-800 k/mm2. We cannot guarantee similar clustering and/or quality for libraries made by customers of the core.
We require that core clients acknowledge the Epigenomics Core of Weill Cornell Medical College
in publications and presentions enabled by Epigenomics core resources.
Sample submission tips
To request a sequencing service, please fill out a request in our
Agilent Crosslab/iLab Service Request LIMS
. Please refer to the
GETTING STARTED
section of our
Sample Workflow
above for detailed sample submission instructions.
Please use the Agilent Crosslab/iLab service ID and the sample number you have indicated in the submission form to label your tubes clearly,
tubes without a service ID and sample number will not be accepted
Once samples are submitted and pass quality control, they are entered into a sample or library queue.
The MiSeq personal sequencer has a single lane and only supports paired end clustering. [
MiSeq PDF
Timeline:
Approximate time for
library preps and sequencing
is
4-8 weeks
if all samples pass quality control.
Approximate time for
data pre-processing and transfer
is
1-2 weeks
from the end of a successful sequencing run.
Illumina Sequencing Coverage Calculator
Pricelist
RRBS (DNA Methylation Sequencing)
Assay Description
educed
epresentation
isulfite
equencing is a modification of the original RRBS protocol (
Gu H. et al. 2011
) and the in-house developed ERRBS method (
Akalin A., Garrett-Bakelman F. et al, 2012
) for base-pair resolution methylation sequencing analysis based on the use of a restriction enzyme to enrich for CpG fragments. RRBS starts with MspI digestion, followed by NGS-library preparation and bisulfite conversion of cytosines.
Our RRBS protocol yields about  ~10% of genomic CpG sites (roughly 3M CpGs in the human genome), and provides enrichment in CpG islands and CpG shores, promoters, exons, introns and intergenic regions (
Garrett-Bakelman F., Sheridan, C. et al. 2015
).
This assay requires RNA-free high molecular weight DNA. Degraded DNA, such as that obtained from FFPE is not suitable.
FFPE samples may be processed using
Agilent's Methylome Capture assay
instead.
Sample Requirement:
Submit 75 ng of genomic DNA,
of molecular weight >20kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Agilent Tape station 4200 or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
100 million (M) read per sample on a paired end read flow cell with 100bp read length (PE100) are recommended for differential methylation analysis, for detailed information please review
Garrett-Bakelman F., Sheridan, C. et al. 2015
Third Party Resource:
RRBS Guide from Babraham Bioinformatics (makers of the Bismark aligner, FASTQC, Seqmonk etc.)
Some studies that have used our RRBS assay:
Odell SC. et al. 2020
Emi T. et al. 2020
Complete Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep
$205
NovaSeq X 200 cycles 1.25B Reads + Bioinformatics processing
$1262
Prices may vary depending on the number of samples.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
RR(Ox)BS (5hmC Sequencing)
Assay Description
educed
epresentation
Ox
idative
isulfite
equencing enables an accurate 5-hydroxymethylcytosine (5hmC) identification.
Please note that the protocol requires two library preparations per sample.
The core uses methodology described in
Akalin A., Garrett-Bakelman F. et al, 2012
and
Garrett-Bakelman F., Sheridan, C. et al. 2015
), with the chemistry from
Tecan’s Ultralow Methyl-Seq with TrueMethyl oxBS
to prepare the libraries.
Sample Requirement:
Submit 400 ng of genomic DNA,
of molecular weight >20kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul. Due to sequencing recipe requirements please submit a
minimum of 4 samples (8 libraries)
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Agilent Tape station 4200 or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
100 million (M) reads per library on a single end read flow cell with 100 sequencing cycles (SR100) are recommended for differential methylation analysis, for detailed information please review
Garrett-Bakelman F., Sheridan, C. et al. 2015
Some studies that have used our RRoxBS assay:
Singh P. et al. 2021
Fortin J. et al. 2023
Complete Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep (per sample)
$255
NextSeq 2000 (400M reads) Single Read 100bp (SR100) + Bioinformatics Processing
$1980
NovaSeq X 100 cycles 1.25B Reads + Bioinformatics Processing
$1742
Prices may vary depending on the number of samples.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
Whole Genome Bisulfite Sequencing (WGBS) - Genome-wide methylation sequencing
Assay Description
Whole genome bisulfite sequencing is the gold-standard approach for comprehensive base-pair resolution and quantitative information at most genomic cytosines. The core uses the
Accel-NGS Methyl-Seq DNA library kit
to prepare libraries. This method uses cytosine bisulfite conversion followed by single stranded ligation of adapters and PCR amplification. [
Application Note
].
Sample Requirement:
Submit 300 ng of genomic DNA,
of molecular weight >20kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Agilent Tape station 4200 or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep
$260
To calculate the sequencing depth required for your desired coverage and species of interest, please use the
Illumina coverage calculator
Estimate pricing as NovaSeq X 300 cycles 1.25B Reads + Bioinformatics processing = $1,345
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
Whole Genome Oxidative Bisulfite Sequencing (WGoxBS) - Genome-wide 5hmC sequencing
Assay Description
hole
enome
Ox
idative
isulfite
equencing (WGoxBS) enables an accurate 5-methylcytosine (5mC) identification and interrogation of both 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC).
Please note that the protocol requires two library preparations per sample, and therefore twice the sequencing depth than WGBS.
The core uses
Tecan’s Ultralow Methyl-Seq with TrueMethyl oxBS
kit to prepare libraries.
Sample Requirement:
Submit 400 ng of genomic DNA,
of molecular weight >20kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul. Due to sequencing recipe requirements please submit a
minimum of 6 samples
(12 libraries).
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Agilent Tape station 4200 or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep
350
To calculate the sequencing depth required for your desired coverage and species of interest, please use the
Illumina coverage calculator
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
Methylome Capture Sequencing (targeted methylome sequencing)
Assay Description
Methylcapture is a hybridization-based approach on platforms containing pre-designed capture oligos, followed by methylation sequencing. There are commercially designed capture libraries available with a range
of epigenetic features that cover ~12% to ~24% of all human genome CpGs [
Agilent SureSelect MethylSeq
Roche CpGiant
].
Please
if you would like to use this service.
Advantages:
Researchers can custom-design capture libraries
that can be used for validation or for discovery of novel epigenomic regions. Please
if you would like to design a custom library.
Amenable for FFPE samples.
Since the technique relies on a sonication step, FFPE DNA can be used.
Sample Requirement:
Depending on the platform used, submit 0.5-2ug of genomic DNA, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2,
RNA-free
and at a concentration of ~50ng/ul
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Perkin Elmer Labchip GX or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep
425
PE100 (2x100 cycles) sequencing is recommended. Depth depends on the chosen commercially available capture panel. To calculate the sequencing depth required for you panel/species, please use the
Illumina coverage calculator
The same data processing pipeline we use for our RRBS assay will be used for methylome capture data as well.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
Enzymatic Methyl-Seq (EMSeq) - Genome-wide methylation sequencing
Assay Description
The
nzymatic
ethyl
Seq
uencing assay provides a high-performance enzyme-based alternative to bisulfite conversion for identification of methylation in low input samples. The core uses the
NEBNext Enzymatic Methyl-seq
kit to prepare libraries.
Sample Requirement:
Submit 10-50 ng of genomic DNA,
of molecular weight >20kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul.
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Agilent Tape station 4200 or agarose gel to determine molecular weight.
Click here for a detailed description of the QC
Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Library Prep
280
To calculate the sequencing depth required for your desired coverage and species of interest, please use the
Illumina coverage calculator
Estimate pricing as NovaSeq X 300 cycles 1.25B Reads + Bioinformatics processing = $1,345
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
5hmC-BIC-Seq (detection of 5-hydroxymethylcytosine modification by affinity pull down)
Assay Description
5hmC-bead-integrated-click-sequencing is an in-house method developed by the core to profile 5hmC containing DNA sequences on a genome-wide scale that uses a novel integrated approach. Typical enrichment protocols use antibodies that recognize a modification or set of modifications on DNA. We chose a covalent chemical labeling technique that could be integrated into the NGS library preparation process. DNA sequences with 5hmC moieties are directly modified with azide-glucose, which can then form a stable biotin conjugate through bio orthogonal click-chemistry. Streptavidin affinity purification enriches 5hmC-containing DNA sequences and integration with sample preparation steps creates a robust assay that can accept limiting levels of input DNA.
Please
if you wish to use this service.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
We process Bisulfite sequencing data using an
in-house
BWA meth
pipeline
to generate methylation calls, related statistics and bam files for the samples.
Sample_CpG.methylKit.gz
Sample_CHH.methylKit.gz
Sample_CHG.methylKit.gz
The Sample_*.methylKit.gz files are tab delimited text files that contain all the reported locations of C's in either CpG, CHG, or CHH, context and their methylation levels.
The minimum read coverage cutoff for this file is 1
cpg.Sample.10x.gz
chh.Sample.10x.gz
chg.Sample.10x.gz
The *.10x.txt.gz files are tab delimited text files that contain the locations of C's in either CpG, CHG, or CHH, context and their methylation levels with a coverage greater than or equal to 10 (>=10x)
Sample_sorted.bam
Sample_sorted.bam.bai
This file contains the complete alignments in binary (BAM) format and a index (.bai) for this BAM file.
Sample_summary.txt
This file summarizes adapter trimming, alignment information, and mapping efficiency of the sample against the genome.
The column headers for the *.methylkit.gz and *.10x.gz are as follows:
chrBase = This is the name (chromosome.base location)
chr = chromosome on which the methylated base is located
base = location of methylated base on the chromosome
strand = forward strand (F) or reverse strand (R)
coverage = read coverage
freqC = % methylated
freqT = % unmethylated
Please refer to the following paper for more information about BWA meth:
Fast and accurate alignment of long bisulfite-seq reads. Pedersen, BS et al. (2014) ArXiv.
ChIPseq (genome-wide mapping of DNA binding proteins)
Assay description
Chromatin Immunoprecipitation Sequencing (Carey et al, 2009
) is the primary method for profiling protein DNA interactions, based on the enrichment of DNA associated to a protein of interest or a histone modification. Despite its increasing use ChIP-seq libraries are among the most challenging to perform and require significant experience and quality control measures. The core offers library preparation for chromatin immunoprecipitated material after successful quality control.
Sample Requirement:
For Input material submit 50 ng at a concentration of 5ng/ul.
For ChIP material submit ~22ng of DNA at a concentration of 0.3ng/ul to 1ng/ul.
Please use a double stranded fluorometric method to determine concentration (Qubit for example); concentrations determined by nanodrop are not reliable for this assay.
Epigenomics Core Quality Control:
QC1 - Quantity:
dsDNA using Qubit Fluorometer.
QC2 - Quality:
High Sensitivity DNA Bioanalyzer chip to determine the spread of the Input chromatin. Accurate representation of the original ChIPd material is obtained when the size range of the DNA fraction required for library preparation (130-230bp) is > 10% of the total DNA provided.
Click here for a detailed description of the QC
Library preparation for this assay is currently done using the IDT-xGen kit.
Some studies that have used our ChIPSeq sequencing service:
Popovic R. et al. 2014
Kuo PY. et al. 2014
Qiao Y. et al. 2013
Complete Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
IDT-xGen ChIP-seq Library Prep
$180
NextSeq 2000 (400M reads) Paired End 50bp (PE50) + Bioinformatics Processing
1980
NovaSeq X 100 cycles 1.25B Reads + Bioinformatics processing
$1742
50M reads of paired end 50 (PE50) sequencing is recommended per sample.
Multiplexing optimization depends on the factor used, for well characterized antibodies or sharp histone marks (for example: H3K4me3), 25 million reads may be sufficient per sample.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are aligned to genomes available via Illumina's iGenome using the
BWA-MEM
aligner. This pipeline results in the following file types:
*.maxL.bam
- The top/best non-filtered alignment for each read in the widely accepted BAM format (a binary version of the
SAM format
) for each sample.
*.merged.bam
- all alignments (including best and multiple alignments) for each read in the widely accepted BAM format (a binary version of the SAM format) for each sample.
*.maxL.bam.bai
*.merged.bam.bai
- Index files (.bai files) for each sample which allow for easier viewing of the bam files in genome browsers such as
UCSC Genome Browser
) or
IGV
).
*-metrics.log
- Summary metrics such as adapter trimming and alignment rates.
ATAC-seq (assay for transposase activity)
Assay description
Assessment of the functional state of chromatin, can be achieved through the digestion of chromatin with Tn5 transposase followed by library preparation and sequencing. The Epigenomics core uses the OMNI-ATAC protocol as detailed in
Corces et al. (Nature Methods, 2017)
Sample Requirement:
After submitting a service request in
Agilent Crosslab
(formerly iLab),
please follow the protocol detailed in this PDF
label the Eppendorf tube with the Agilent CrossLab ID and the sample number (as in the service request) and bring samples after tagmentation to room A-427
. We do not accept cells for ATAC-seq.
Instructions for preparation of OMNI ATAC-seq samples [PDF]
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
ATAC-seq (Omni) Library Prep (Tagmented Samples)
$105
NovaSeq X 100 cycles 1.25B Reads + Bioinformatics processing
$996
50M reads of paired end 50 (PE50) sequencing is recommended per sample.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are aligned to genomes available via Illumina's iGenome using the
BWA-MEM
aligner. This pipeline results in the following file types:
*.maxL.bam
- The top/best non-filtered alignment for each read in the widely accepted BAM format (a binary version of the
SAM format
) for each sample.
*.merged.bam
- all alignments (including best and multiple alignments) for each read in the widely accepted BAM format (a binary version of the SAM format) for each sample.
*.maxL.bam.bai
*.merged.bam.bai
- Index files (.bai files) for each sample which allow for easier viewing of the bam files in genome browsers such as
UCSC Genome Browser
) or
IGV
).
*-metrics.log
- Summary metrics such as adapter trimming and alignment rates.
Exome Capture
Assay description
The Agilent SureSelect platform is a solution-based system using 120-mer (biotinylated cRNA baits) to capture regions of interest, enriching them out of a NGS sonicated genomic library. Targeted regions can be interrogated for the purpose of identifying structural rearrangements and SNPs. [
SureSelect PDF
Sample Requirement:
Please submit a minimum of 12 samples.
Submit 3ug of purified DNA, Nanodrop A260/280 ratio> 1.7; A260/230 =2.0-2.2
Epigenomics Core Quality Control:
dsDNA using Qubit Fluorometer, 0.8 % agarose gel to determine quality
Complete Pricelist / Multiplexing Calculator
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
External Academic Price
Exome Capture TruSeq Compatible Library Prep
528
600
HiSeq PE75 Paired End Clustering and 3 x 50 Sequencing Cycles
2050
3000
1 lane of PE75 sequencing is recommended for commercially available capture libraries. To calculate the sequencing depth required for you species of interest, please use the
Illumina coverage calculator
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are aligned to genomes available via Illumina's iGenome using the
BWA-MEM
aligner. This pipeline results in the following file types:
*.maxL.bam
- The top/best non-filtered alignment for each read in the widely accepted BAM format (a binary version of the
SAM format
) for each sample.
*.merged.bam
- all alignments (including best and multiple alignments) for each read in the widely accepted BAM format (a binary version of the SAM format) for each sample.
*.maxL.bam.bai
*.merged.bam.bai
- Index files (.bai files) for each sample which allow for easier viewing of the bam files in genome browsers such as
UCSC Genome Browser
) or
IGV
).
*-metrics.log
- Summary metrics such as adapter trimming and alignment rates.
DNASeq (whole genome sequencing)
The Epigenomics Core will be discontinuing this service starting Sep 15th, 2018.
To continue availing of this service, please contact
Dr. Jenny Xiang
at the Genomics Resources Core Facility (GRCF).
GRCF Service Request Portal
An entire genome can be sequenced allowing SNP discovery, identification of copy number variations and chromosomal rearrangements [
Nanokit
Sample Requirement:
Submit 250 ng of genomic DNA,
of molecular weight >40kb
, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2;
RNA-free
and at a concentration of ~20ng/ul
Epigenomics Core Quality Control:
dsDNA using Qubit Fluorometer, 0.8 % agarose gel to determine quality
Click here for a detailed description of the QC
Pricelist
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
TruSeq DNA-seq Library Prep
$300
Two lanes of PE75 or PE100 sequencing recommended for human DNAseq. To calculate depth required for your custom application, please use the
Illumina coverage calculator
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are aligned to genomes available via Illumina's iGenome using the
BWA-MEM
aligner. This pipeline results in the following file types:
*.maxL.bam
- The top/best non-filtered alignment for each read in the widely accepted BAM format (a binary version of the
SAM format
) for each sample.
*.merged.bam
- all alignments (including best and multiple alignments) for each read in the widely accepted BAM format (a binary version of the SAM format) for each sample.
*.maxL.bam.bai
*.merged.bam.bai
- Index files (.bai files) for each sample which allow for easier viewing of the bam files in genome browsers such as
UCSC Genome Browser
) or
IGV
).
*-metrics.log
- Summary metrics such as adapter trimming and alignment rates.
Targeted Resequencing (interrogation of genes specific for heme malignancies)
Assay description
Using Raindance’s single-molecule pico droplet PCR reaction three panels that contain genes specific for heme malignancies have been designed:
Myeloid leukemia
Chronic Lymphocytic Leukemia
Lymphoma
This is a high-throughput technique, a minimum of 40 samples are required.
Sample Requirement:
Submit 500 ng of genomic DNA, of molecular weight >40kb, Nanodrop A260/280 ratio >1.7; A260/230=2.0-2.2; at ~20ng/ul
Epigenomics Core Quality Control:
Determination of concentration of double stranded DNA (dsDNA) using Qubit Fluorometer, Perkin Elmer Labchip GX or agarose gel to determine molecular weight.
Complete Pricelist / Multiplexing Calculator
Service
Internal
(WCM, WCM Qatar & Cornell U) Price
External Academic Price
Raindance Library Prep
200
400
MiSeq
600
Sequencing Cycles v3
1500
2400
Please
if you wish to use this service.
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are aligned to genomes available via Illumina's iGenome using the
BWA-MEM
aligner. This pipeline results in the following file types:
*.maxL.bam
- The top/best non-filtered alignment for each read in the widely accepted BAM format (a binary version of the
SAM format
) for each sample.
*.merged.bam
- all alignments (including best and multiple alignments) for each read in the widely accepted BAM format (a binary version of the SAM format) for each sample.
*.maxL.bam.bai
*.merged.bam.bai
- Index files (.bai files) for each sample which allow for easier viewing of the bam files in genome browsers such as
UCSC Genome Browser
) or
IGV
).
*-metrics.log
- Summary metrics such as adapter trimming and alignment rates.
Single Cell Gene Expression
Assay description
Our single-cell gene expression services includes the following 10x Genomics
GEM-X
technology
single cell gene expression
assays [
PDF
]:
single-plex
On-Chip-Multiplexing (OCM)
and
Flex
Single cell assays require prior consultation and coordination with the core.
Please contact us at
epicore-sc-coord@med.cornell.edu
at least three weeks before dropping off your samples to schedule a time. We currently only accept external customers coming from within a mile radius of our facility.
The 10x Genomics Chromium Single Cell Expression Solution
provides high-throughput, single cell expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cell types. This is also available in conjunction with
Feature Barcoding
(Cell Surface Protein, CRISPR Screening or Custom).
The protocol requires a suspension of viable single cells as input.
Minimizing the presence of cellular aggregates, dead cells, non-cellular nucleic acids and potential inhibitors of reverse transcription is critical to obtaining high quality data.
Sample Requirement:
The total number of cells required in the suspension used as input is determined by the desired cell recovery target (between 500-20000 cells for single-plex and 500-5000 individual cells for OCM); required sequencing read depth depends on the desired cell target. Please check user guides
CG000731
and
CG000768
for details. [
Library Prep User Guides
For Flex we require 300,000 to 1,000,000 fixed cells. Please check user guide
CG000478
Given the variety of cells and sample types, general guidelines for sample preparation need to be optimized by each customer. Please check the
10X Genomics Single Cell Gene Expression Sample Prep Guide
for information on how to prepare cells.
Singleplex Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Multiplex Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Single Cell 3P Library Prep (1 sample)
$2400
On Chip Multiplexing (OCM), (4 samples), each
$895
Single Cell 3P Library Prep (2-4 samples), each
$2150
Flex v2 Gene Expression (12 or 16 samples), each
$600
Single Cell 3P Library Prep (>5 samples), each
$2050
Flex v2 Gene Expression (4 or 8 samples), each
$1219
Single Cell 3P Gene expression with Cell Hashing Library Prep (per sample)
$2550
Additional Feature Barcoding Library Prep (per sample)
$250
GEM-X Chip
$325
GEM-X Chip
$325
NovaSeq X (100 cycles, 1.25B reads) + Bioinformatics Processing
$996
We sequence Chromium single cell samples on a pair end flow cell (28-10-10-90) with a 2x50 cycles kit.
Chromium recommends a minimum of 20,000 reads per cell for gene expression
(Example: Samples containing from 2000-10,000 cells would require from 40-200 million reads per sample). Please contact us at
epigenomicscore@med.cornell.edu
to discuss the appropriate number of reads required for your experiments.
Data Processing
The sequencing data will be demultiplexed and post-processed using
custom pipelines provided by 10x Genomics
Feature Barcoding (Cell Surface Protein, CRISPR Screening or Custom) and Cell Multiplexing assays require additional information for post-processing with cellranger
(for collating the feature counts with the gene expression counts).
Below please find a description of the columns in the Feature Reference table:
Sample Name
The name you have used for the samples while submitting them in iLab
Feature Name
Human-readable name for this feature.
Must not contain whitespace.
This name will be displayed in Loupe Cell Browser.
Read (R1 or R2)
Specifies which RNA sequencing read contains the Feature Barocde sequence. Must be R1 or R2. Note: in most cases R2 is the correct read.
Pattern / Position of Barcode
The pattern column can be made up of a combination of these elements:
5P: denotes the beginning of the read sequence.
May appear 0 or 1 times, and must be at the beginning of the pattern. Only 5P or 3P may appear, not both.
3P: denotes the end of the read sequence.
May appear 0 or 1 times, and must be at the end of the pattern.
N: denotes an arbitrary base.
A, C, G, T: denotes a fixed base that must match the read sequence exactly.
(BC): denotes the Feature Barcode sequence as specified in the sequence column of the feature reference.
Must appear exactly once in the pattern.
Sequence
Nucleotide barcode sequence associated with this feature. E.g., antibody barcode or sgRNA protospacer sequence.
The
10X Genomics Feature Barcoding page
has detailed information about the post processing of feature barcoding data and required input for cellranger.
Single Cell Immune Profiling
Assay description
Our single-cell gene expression services includes the following 10x Genomics
GEM-X
technology
single cell gene expression
assays [
PDF
]:
Single plex
and
On-Chip-Multiplexing (OCM)
Single cell assays require prior consultation and coordination with the core.
Please contact us at
epicore-sc-coord@med.cornell.edu
at least three weeks before dropping off your samples to schedule a time. We currently only accept external customers coming from within a mile radius of our facility.
The Chromium Single Cell Immune Profiling Solution
The Chromium Single Cell Immune Profiling Solution is a comprehensive approach to simultaneously examine the cellular context of the adaptive immune response and immune repertoires of hundreds to tens of thousands of T and B cells in human or mouse on a cell-by-cell basis. With the addition of Feature Barcoding Technology, you can now also detect and analyze additional cellular readouts such as cell surface markers and antigen specificity to enhance immune cell phenotyping and study dynamic interactions between lymphocytes and target cells.
Sample Requirement:
The protocol requires a suspension of viable single cells as input.
Minimizing the presence of cellular aggregates, dead cells, non-cellular nucleic acids and potential inhibitors of reverse transcription is critical to obtaining high quality data.
The total number of cells required in the suspension used as input is determined by the desired cell recovery target (between 500-20000 cells for single-plex and 500-5000 individual cells for OCM); required sequencing read depth depends on the desired cell target. Please check user guides
CG000733
and
CG000770
for details. [
Library Prep User Guide
Given the variety of cells and sample types, general guidelines for sample preparation need to be optimized by each customer. Please check the
10X Genomics Single Cell Immune Profiling Sample Prep Guide
for information on how to prepare cells.
We can process up to 4 samples at a time.
The library preparation prices below are for the 5 prime gene expression assay as well as either a T or a B cell V(D)J assay.
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Single Cell 5P gene expression + VDJ repertoire (1 sample)
$2500
On Chip Multiplexing (OCM), 4 samples, each
$895
Single Cell 5P gene expression + VDJ repertoire (2-4 samples)
$2200
Single Cell 5P gene expression + VDJ repertoire (>5 samples)
$2100
Single Cell 5P gene expression + VDJ repertoire + Hashing
$2800
Additional Immuno Profiling Feature Barcoding Library Prep (per sample)
$250
GEM-X Chip
$325
NovaSeq (100 cycles 1.25B Reads) + Bioinformatics Processing
$996
We sequence Chromium single cell samples on a pair end flow cell (28-10-10-91) with a 2x50 cycles kit.
Chromium recommends 50,000 reads per cell with a minimum of 20,000 reads per cell for gene expression
(Example: Samples containing from 2000-10,000 cells would require from 100-500 million reads per sample).
The T or B cell specific V(D)J assay requires 5000 reads per cell.
Please contact us at
epigenomicscore@med.cornell.edu
to discuss the appropriate number of reads required for your experiments.
Data Processing
The sequencing data will be demultiplexed and post-processed using
custom pipelines provided by 10x Genomics
Single Cell Epigenomics
Assay description
We are currently offering
Single Cell ATAC
(NextGEM) as well as
Epi Multiome ATAC + Gene Expression
Single cell assays require prior consultation and coordination with the core.
Please contact us at
epicore-sc-coord@med.cornell.edu
at least three weeks before dropping off your samples to schedule a time.
The Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin) Solution
The Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin) Solution accelerates the understanding of the regulatory landscape of the genome, thereby providing insights into cell variability. The high-throughput chromatin profiling of single cells in parallel allows researchers to see how chromatin compaction and DNA-binding proteins regulate gene expression at high resolution.
Sample Requirement:
The total number of nuclei required in the suspension used as input is determined by the desired cell recovery target (between 500-10,000 cells); required sequencing read depth depends on the desired cell target.
Chromium recommends 25,000 reads per nuclei
(Example: 4 samples at 10,000 cells can be sequenced on one NovaSeq6000 SP flowcell, sequencing recipe 50-8-16-50)
Given the variety of cells and sample types, general guidelines for sample preparation need to be optimized by each customer. Please check the relevant
Sample Prep Guides
for information on how to prepare cells.
We can process up to 4 samples at a time and can only accept samples from local/internal clients.
Service
Internal Price
Single Cell Multiome ATACseq & Gene Expression Library Prep
$4000
Single Cell ATAC Library Prep
$2412
Next GEM Chip J
$320
Next GEM Chip H
$325
NovaSeq X (100 cycles, 1.25B reads) + Bioinformatics Processing
$996
Data Processing
The sequencing data from Chromium Single Cell ATAC will be demultiplexed and post-processed using
custom pipelines provided by 10x Genomics
The sequencing data from our Single Cell RRBS assays will be demultiplexed and post-processed in the same manner as our RRBS samples. Please click on the ERRBS section in the right hand menu for details.
Chromium Long Range Sequencing
Assay description
We are currently offering
10x Genomics applications
for our long range genomic assay.
The Chromium Long Range Genome Solution
provides long range information on a genome-wide scale, including variant calling, phasing and extensive characterization of genomic structure. Applications include interrogating heterogeneous cell populations, resolving phasing information and detecting structural variations. Please contact us at
epigenomicscore@med.cornell.edu
for further information and to discuss the details of your project.
Data Processing
The sequencing data will be demultiplexed and post-processed using
custom pipelines provided by 10x Genomics
Spatial Transcriptomic Profiling
Assay Description
We are currently offering
Visium CytAssist Spatial Transcriptomics
, H&E workflow for mouse and human tissue samples. This assay allows mapping of the transcriptome within morphological context.
Spatial Transcriptomics assays require prior consultation and coordination with the core.
Please contact us at
epigenomicscore@med.cornell.edu
to discuss your project.
Sample Requirements
RNA quality control (recommended DV200 > 30% for FFPE, DV200 > 50% for FxF, RIN > 4 for FF, RIN > 7 for FF HD 3’)
A preliminary H&E staining should be performed to determine the region of interest.
Samples can be FFPE, FF, or FxF tissue sections mounted on recommended histology slides (Tissue Slides). For a standardized workflow at the Epigenomics Core, we use Fisherbrand SuperFrost Plus slides (Cat# 12-550-15), while Schott Nexterion Slide H - 3D Hydrogel Coated (PN-1800434) is preferred for TMAs or tissues prone to detachment. Other validated options are listed in the 10x Genomics planner files CG000556 (SD), CG000698 (HD), and CG000803 (HD 3’). Note that the Visium HD 3’ workflow is restricted to FF samples.
The region of interest (6.5x6.5mm or 11x11mm) must be within an allowable area on the Tissue Slide.
For sections thickness, sectioning protocol, tissue placement, size and location of the allowable area on the slide, tissue slides drying, storing and transfer to the Epigenomics Core, please refer to the
Visium CytAssist Spatial Transcriptomics [PDF] infographic guide
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
Visium CytAssist SD 6.5x6.5mm (library prep for two slide capture areas)
$4700
Visium CytAssist SD 11x11mm (library prep for two slide capture areas)
$9021
Visium CytAssist HD (library prep for two slide capture areas)
$8750
Visium CytAssist 3' HD (library prep for two slide capture areas)
$9000
NovaSeq X (100 cycles, 1.25B reads) + Bioinformatics Processing
$996
Submit a minimum of 2 tissue slides, or multiples of 2. Multiple tissues may be placed on a tissue slide, provided they fit within the 6.5x6.5mm (or 11x11mm) “Region of Interest” area.
Data Processing
The sequencing data from Visium Spatial Gene Expression will be demultiplexed and post-processed along with microscope generated images using the
spaceranger software provided by 10x Genomics
Single Cell Spatial Profiling
Assay Description
We are currently offering multiomic single-cell spatial profiling via the
CosMx Spatial Molecule Imager from NanoString
for FFPE and Fresh Frozen samples/slides. This assay allows high plex profiling of transcriptomics and proteomics within morphological context at sub-cellular resolution.
Single cell spatial profiling assays require prior consultation and coordination with the core.
Please contact us at
epicore-sc-coord@med.cornell.edu
at least three weeks before dropping off your samples to schedule a time.
Sample Requirements
FFPE microtome (RNA and Protein) or fresh frozen (RNA) sections on Nanostring-validated positively charged histology slides are required.
Please review this detailed PDF to plan your CosMx experiment
Preliminary sectioning, and H&E staining on parallel sections are required to determine the regions of interest and assign the prospective “Field of View” (FOVs) to be scored. Each “Field of View” (FOV) corresponds to a single CosMx 23x/1.1 NA objective acquisition area. FOVs are square-shaped, with the sides parallel to the slide edges. (It is not possible to rotate the FOVs to better fit the regions of interest.)
CosMx Planning Checklist [PDF]
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
1000x panel, 150 FOVs per slides, on a 2 slide run. Price for each slide
$5396
CosMx 1000x panel, 300 FOVs per slides, on a 2 slide run. Price for each slide
$5996
CosMx 1000x panel, 300 FOVs per slides, on a 4 slide run. Price for each slide
$4650
CosMx 6000x panel, 150 FOVs, Price for each slide
$8637
CosMx WTX (human) panel, 150 FOVs per slide on a 2 slide run
$19000
CosMx-Protein 64x plex, 150 FOVs per slide on a 2 slide run. Price for each slide
$4126
CosMx-Protein 64x plex, 300 FOVs per slide on a 2 slide run. Price for each slide
$4330
Submit a minimum of 2 histology slides, or multiples of 2.
Data Processing
CosMx spatial imaging data will be post-processed via the
AtoMx Spatial Informatics Platform (SIP)
provided by Bruker (Nanostring). Cell segmentation and Foundational pipelines that normalize, dimensionally reduce, and perform recommended downstream analysis will be run in AtoMx and you will get access to view, explore, and/or export your data via this SIP. Please refer to the
CosMx Data Analysis Manual
for further details about the post-processing and export of your CosMx/AtoMx data.
We will also share the following files (convenient for consumption by third-party tools) via our data portal PubShare:
Seurat Object
- The .rds file is a
Serurat Object
(that includes polygon coordinates, metadata and dimensional reduction outputs).
Flat Files
- The .tgz file is a tar-gzipped archive of flat files from the AtoMx pipeline export. Included flat files are as follows:
Count matrix file (cells with gene counts)
Cell metadata file (local (within the FOV)/global (within the flow cell) cell coordinates, morphology marker intensities)
Global transcripts file (global coordinates for every individual transcript on the flow cell; not applicable for protein studies)
Global cell boundaries file (global coordinates for the vertices of the polygon that represents the cells' boundaries - a representation of the cell segmentation)
Global FOV position file (coordinates of each FOV center)
Please note that your Pubshare account is separate from your Agilent iLab account (used to create your service request) as well as your account to access the AtoMx SIP.
If you don't already have access to the AtoMx SIP, expect an email from Okta (noreply@okta.com) to set up your account for AtoMx.
Please be advised that your AtoMx account set-up link from Okta expires a week after it is sent.
RNASeq (transcriptome analysis)
The Epigenomics Core will be discontinuing this service starting Sep 15th, 2018.
To continue availing of this service, please contact
Dr. Jenny Xiang
at the Genomics Resources Core Facility (GRCF).
GRCF Service Request Portal
To understand the dynamic state of the cell transcriptome, the core provides a range of RNA sequencing library preparations that can assess total RNA, mRNA and noncoding RNA
in higher eukaryotes
TruSeq Stranded mRNA:
polyA selection followed by library preparation is the standard and most widely used method for quantifying mRNA expression. Our protocol retains the strand orientation of the transcripts. [
MORE
Sample Requirement:
Submit a minimum of 250 ng of purified RNA, at a concentration of 50 ng/ul; A260/280 > 2, picogreen quantification or bioanalyzer trace, RNA integrity number (RIN) >8.0
Epigenomics Core Quality Control:
RNA Nano Bioanalyzer chip to determine RIN number and picogreen quantification.
Ultra Low Input RNA:
mRNA profiling of very low amounts of input material (<20ng) that is based on polyA selection using the
Clontech SMArter technology
. Often used for clinical samples or expression profiling of few cells (e.g. neurons, stem cells).
Sample Requirement:
Submit a minimum of 20 ng of purified RNA, at a concentration of 5 ng/ul; A260/280 > 2, picogreen quantification or bioanalyzer trace, RNA integrity number (RIN) >8.0 OR 1000 cells in 10ul of Clontech 1X Single Cell Lysis buffer (cat # 635013)
Epigenomics Core Quality Control:
RNA Pico Bioanalyzer chip to determine RIN number and picogreen quantification.
TruSeq Stranded Total RNA:
rRNA depletion followed by strand-specific library preparation. Generally more applicable towards simultaneous profiling of non-coding transcripts and mRNA although requires more input material. [
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Sample Requirement:
Submit a minimum of 250 ng of purified RNA, at a concentration of 50 ng/ul; A260/280 > 2, picogreen quantification or bioanalyzer trace, RNAs with an RNA integrity number (RIN) < 8 can be used
Epigenomics Core Quality Control:
RNA Nano Bioanalyzer chip and picogreen quantification.
TruSeq RNA [DISCONTINUED]:
Please use TruSeq-stranded mRNA instead.
Agilent's Guide to Interpreting Bioanalyzer Results
Some studies that have used our RNASeq service:
Kuo PY. et al. 2014
Satyaki PR. et al. 2014
Pimentel H. et al. 2014
Complete Pricelist / Multiplexing Calculator
Service
Internal Price
(WCM, WCM Qatar & Cornell U)
External Academic Price
TruSeq RNA-seq (polyA) Library Prep
185
300
TruSeq Stranded RNA-seq (polyA) Library Prep
170
225
Ultra Low Input RNA-seq (polyA) Library Prep
280
370
TruSeq Total RNA-seq (stranded) Library Prep
300
400
HiSeq SR50 Single Read Clustering and 1 x 50 Sequencing Cycles
1100
1600
HiSeq PE50 Paired End Clustering and 2 x 50 Sequencing Cycles
1650
2200
Sequencing depth required depends on your application.
For human transcriptome differential gene expression we recommend 4 samples per lane of SR50 sequencing.
For rare splicing events, translocations and some other experiments PE50 sequencing is recommended.
To calculate depth required for your custom application, please use the
Illumina coverage calculator
Data Processing
Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed and converted to
FASTQ
files using the Illumina
bcl2fastq
software.
Your raw data will be available for download as a tar compressed archive (Sample_*.tar) of gzipped FASTQ files for each sample. Raw data can be post-processed upon request.
FASTQ files generated as described above are adapter trimmed and aligned to genomes available via Illumina's iGenome using the
STAR
aligner. Only raw reads that pass Illumina's purity filter are aligned. This pipeline results in the following file types:
*.bam
- Upon alignment (if requested) the aligned data processed by
STAR
aligner is in the widely accepted BAM format (a binary version of the
SAM format
).
*.bai
- This is an index file for your BAM alignments and allows certain browsers (such as the
IGV
browser) to better view the .bam file.
*-SJ.out.tab
- The high confidence collapsed splice junctions in tab-delimited format. Only junctions supported by uniquely mapping reads are reported.
*-Log.final.out
- A text file containing the STAR aligner generated summary statistics for the alignment of each sample.
Bioinformatics Support
The Epigenomics Core Facility provides
data analysis services and consultation
on a per project basis. These include but are not limited to:
RNASeq differential expression
RRBS differential methylation
ChIPSeq peak calling and differential binding
Functional annotation and analysis
Single Cell and Spatial Profiling
Custom alignments
We require that publications and/or presentations enabled by custom data analysis through core personnel contain proper author credit for the bioinformatician. Please discuss this with us before publication/presentation.
Please note that
some bioinformatics support is included in the price for some of our services
For example:
Quality control statistics for your samples
2 years data storage on our backed up servers with unlimited password protected access for sequencing projects
Illumina iGenome alignments (if available and when requested for internal customers)
Methylation calling for DNA methylation sequencing projects (where genomes are available)
Methylation ratios for Epityper mass array projects
Splice junction tables for RNASeq alignments
Please review the
Data Analysis and Retrieval section
of our
Frequently Asked Questions page
for further details.
Pricelist
Service Type
Internal Price (WCM, WCM Qatar & Cornell U)
External Academic Price
Data analysis support / hour
$125
$175
Third Party Analysis Resources
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