Services
The Advanced Technology Genomic Core (ATGC) offers a variety of services, including:
Additional Services
The ATGC offers several additional services, including:
- Droplet digital PCR
- Fluorescent fragment analysis
- nanoString nCounter analysis
- Bionano Optical Genome Mapping
Droplet Digital PCR
Droplet Digital PCR (ddPCR) is used to perform a variety of analyses, including absolute quantification of nucleic acid targets, gene expression or rare event (mutation) detection. The Bio-Rad QX200 system uses fluorescence (probe or EvaGreen) and water-oil emulsion droplets, approximately 1 nanoliter in size, to isolate and analyze the targets in individual droplets. This technology provides very sensitive analysis and is well-suited for detection of low frequency targets.
View the service pricing schedule for more information about ddPCR pricing.
Droplet Digital PCR applications
- Detect copy number variations without the use of a standard curve
- Detect rare mutations or sequences- detect one mutant in a background of up to 2,000 wild-type molecules
- Perform gene expression and microRNA analysis
- Perform single cell analysis
- Detect pathogens and analyze the microbiome
- Quantitate NGS libraries and validate NGS results
Instrumentation
The Bio-Rad QX200 ddPCR system consists of a droplet generator to form approximately 20,000 water-oil emulsion droplets and a droplet reader to count the targets in each droplet.
How the ATGC’s ddPCR service works
- Investigators are responsible for purchasing custom or inventoried (validated) assays and the appropriate master mix directly from Bio-Rad: http://www.bio-rad.com/en-us/product/primepcr-pcr-primers-assays-arrays.
- The investigator schedules an instrument run with the ATGC.
- The investigator sets up the Eva Green or TaqMan-style, probe-based reactions in a 96-well plate (Bio-Rad P/N 12001925 preferred) with a total volume of 22ul. If the reactions use EvaGreen as a fluorophore, the reactions must be prepared in a Bio-Rad 96-well PCR plate (P/N 12001925).
- The investigator delivers the reactions in a sealed 96-well plate to the ATGC’s ddPCR service.
- The ATGC generates the droplets from the investigator's reactions.
- Samples are transferred from the droplet generator to a 96-well Bio-Rad plate, sealed and placed in a thermal cycler for PCR.
- Following PCR amplification the samples are placed on the Droplet Reader where the droplets are examined sequentially, similar to a flow cytometer, providing an independent digital measurement. The droplets are scored as either positive or negative for the marker of interest.
- Data Output-Investigators are provided with an analysis file. Further analysis of data can performed, by the investigator, using Bio-Rad’s free QuantaSoft™ Software which is free to download here: http://www.bio-rad.com/en-us/SearchResults?Text=quantasoft&TabName=SOFTWARETYPE
Droplet Digital PCR submission
- Contact Denaha (D.J.) Doss (DJDoss@mdanderson.org) to schedule your instrument run. Please give at least 24 hour notice.
- Download and complete the Service Request and Plate Layout forms and email them to: ddPCRSubmissions@mdanderson.org. You may also contact the ATGC for the forms.
- Provide the reactions in a 96-well plate (Bio-Rad P/N 12001925 preferred), sealed and centrifuged.
- Results can be expected the same day, provided the plate is submitted before noon.
ATGC contact information
Denaha (D.J.) Doss
Email: djdoss@mdanderson.org
Erika Thompson
Email: ejthomps@mdanderson.org
Bio-Rad support for assay design or data analysis
By telephone: 1-800-4-BIORAD (1-800-424-6723)
By email: support@bio-rad.com
Fluorescent Fragment Analysis
Fluorescent Fragment Length Analysis is used to determine genotypes and assess loss of heterozygosity and microsatellite instability. DNA Fragment Analysis permits a range of applications, such as patient
sample authentication with regards to cell lines and detection of aneuploidy. This technique involves using fluorescent primers during amplification to label PCR products. These products are then separated by capillary electrophoresis. Users are responsible for providing fluorescently-labeled PCR products which are run together with sizing standards on the 3730 Genetic Analyzer. Attributes like detection of multi-repeat sections of genome, a quick turnaround time, with a greater precision and resolution facilitate scientists to select fragment Analysis. Fragment Analysis can investigate more than 20 loci in a single run. Multiplexing capability; and alleles for overlapping loci individualized by locus-specific primers with distinctive chromophores makes the technique of Fragment Analysis lucrative and cost-effective.
View the service pricing schedule for more information about fluorescent fragment analysis pricing.
Instrumentation
The facility performs this analysis on the 3730 capillary platform. Running time is two hours for 96 samples up to 750 bases in size. The ABI (Thermo Scientific) GeneScan™ platform has several advantages over conventional methods. These include running individual lane sizing standards so lane-to-lane migration variation is eliminated. This instrument also provides increased sensitivity of detection, which allows the use of less DNA. This can be important when working with limited tumor samples. Also, PCR-based tests are easy to standardize and automate so the results are very reproducible.
The optimum size for this kind of analysis is 100-400 bases. The size standard used for the analysis is LIZ 500. Please do not label your primers with LIZ dyes.
Steps for fluorescent fragment analysis
1. Guidelines for good primer design:
- Design forward and reverse primers so primers have comparable Tm.
- BLAST primer sequence to make sure there is only one target for the primer.
- Design the reverse primer with a tail. This is very important in making accurate allele calls as taq polymerase randomly incorporates an adenosine at the 3' end of the template during PCR. This is frequently referred to as the 'Plus A' phenomenon. One way to force a “+A” reaction to completion will be to add a GTTCTT tail on the 5’ end of the reverse primer .
- This will reduce difficult data interpretation that is caused by trying to discriminate the +A allele from the true allele.
- Also adding a 30-minute final extension at 70˚C at the end of PCR will promote the completion of 'Plus A' addition.
2. Optimize PCR by performing a magnesium chloride titration and varying the annealing temperature.
3. Quantitate the PCR product. As little as 0.5-1 ng of product will give a strong signal. It is also important to remember that too much product will cause the CCD camera to saturate, and the software will no longer be able to correct for spectral overlap. This will result in what is known as 'pull-up peaks'.
Critical fluorescent fragment analysis factors
The following factors are crucial to the success of detecting small mobility factors:
- Primer labeling: Life Technologies (Thermo Scientific) recommends using 5' end-labeled primers. Please label only ONE of your primers.
- Size standard: An internal standard that ranges from 35-500 bp is used in the reactions. It is strongly recommend that PCR products provided be between 50-500 bp in size. The internal standard used by the software to analyze the fragment lengths is labeled with LIZ (orange). Therefore, it is not recommended to label the primer to synthesize the PCR products with LIZ.
- Control DNA: We also recommend that you submit PCR product from an individual with a known genotype. This will help serve as a troubleshooting device for the customer as well as the service. It allows for monitoring gel-to-gel variability as well as providing information on the effectiveness of the PCR amplification.
Fluorescent fragment analysis FAQs
What is GeneMapper™?
This is the software that allows for the accurate determination of the length of fluorescently labeled PCR fragments. This technology will allow multiplexing of many different fragments in one lane and so allow for rapid screening of multiple loci. The software can be used to determine the size, height and area under the allele peak. This information can then be used to determine the size of the STR at a particular allele, whether it shows microsatellite instability or whether an allele has been lost.
What dyes can I use?
Options for primer labels include 6FAM, NED, VIC or PET. The common multiplexes done in this laboratory are NED, 6FAM and VIC. Only one primer should be fluorescently labeled. Primers should be labeled at the 5' end of the primer. Custom kits and pre-optimized kits are available from Life Technologies.
What are Microsatellites?
Microsatellites are highly informative markers found in the genome. They are defined as tandem repeats of two- to 10-bp units and may be present as perfect or imperfect repeats, e.g., CAGCAGCAGCAG. Repetitive regions are highly polymorphic across populations but tend to be conserved within an individual and their family and, therefore, act as informative molecular markers. The number of repeats found in each individual are highly variable with as few as two, or as many as 50, copies in each microsatellite unit. The most commonly used microsatellite markers are dinucleotide, trinucleotide and tetranucleotide repeats. These genetic markers can be used as valuable tools in researching the fields of molecular population genetics, medical genetics, forensic DNA research and evolutionary biology.
Primers are constructed from the DNA flanking microsatellite regions as the adjacent DNA is usually conserved. During PCR the regions containing the microsatellite are amplified. The PCR products that are fluorescently labeled are then separated by size using capillary or gel electrophoresis. The size of the PCR product can then be used to identify the number of repeats.
What is Multiplexing?
When individual PCR reactions are each labeled with a different dye and mixed prior to running on the instrument, many PCR products can be run and sized in one lane. This allows for high throughput microsatellite screening. In this facility as many as five independently amplified PCR products have been multiplexed successfully. Commercial kits are available with which as many as 10-16 loci can be multiplexed. We strongly recommend that you consider multiplexing when submitting a large number of samples.
A second option for multiplexing involves mixing multiple primer sets with one template and simultaneously co-amplifying all the products. This requires a lot more optimization, as all the markers may not amplify with equal efficiency during a reaction.
What are Pull-Up Peaks?
When the signal from the PCR products is too high, the instrument software can no longer correct for the spectral overlap that exists for a dye set. Ideal rfu values should remain between 200-2000. This means that other colored small peaks will appear under the position of one strong peak. This will create errors in data interpretation. PCR products submitted for this service should have concentrations of 0.5-1 ng/µl.
Procedure for sample submission:
1. Customers need to submit their purified, fluorescent-labeled PCR products at the concentration of 0.5 to 1ng /ul, in either 0.5ml tubes or in a 96-well PCR plate. Most commonly used dyes for PCR reactions are 6FAM, VIC, NED and PET.
2. Customers need to send an excel sheet with sample names and the dye used in PCR reactions, to Ms. Santasri Sen (santsen1@mdanderson.org).
3. A completed billing form including customer’s account information will also need to be sent to Ms. Sen. The form may be downloaded from the core facility web site.
4. The facility runs an optimization plate for first time users, running each sample in different dilutions to determine the correct dilution to produce the best possible results, and to avoid pull up peaks. QC data will be sent to customers.
5. Turnaround time is usually 24 to 48 hours.
6. Customers can download Peak Scanner software, available free from Applied Biosystems website , to interpret data. The software enables to identify peaks and sizing them.
nanoString nCounter Analysis
The nanoString nCounter Analysis system utilizes a novel fluorescent color-coded molecular barcode technology coupled with single molecule imaging to perform digital nucleic acid (RNA and DNA) counting. This technology enables investigators to profile hundreds of targets simultaneously, up to 800 mRNA or miRNA targets in a single reaction for many kits.
View the service pricing schedule for more information about nanoString nCounter analysis pricing.
System highlights and applications
- Multiplex up to 800 targets in a single reaction (certain assays)
- Minimal/No amplification or enzymatic reactions
- Utilize small sample quantities (100 ng RNA, 300 ng gDNA), contact D.J. Doss for specific kit requirements
- Diverse and difficult sample types: blood, tissue and FFPE derived samples, ChIP DNA
- High sensitivity and reproducibility
- Fast turnaround, sometimes as little as 4 working days
RNA applications include:
- Targeted analysis of complex gene expression networks.
- Characterization of gene fusions and splice variants.
- miRNA expression analysis.
- miRGE analysis (miRNA and mRNA at the same time).
- LncRNA expression analysis (custom order).
DNA applications include:
- Validation of Illumina NGS data.
- Copy number variation analysis.
- ChIP screening (ChIP-String).
Instrumentation
The nCounter MAX/FLEX system consists of a robotic nCounter Prep Station for sample processing and the nCounter Digital Analyzer for collecting data.
Sample submission
NanoString offers a variety of pre-designed, off-the-shelf assays*. In addition, investigators can work with nanoString to supplement existing panels with custom probe-sets or design complete custom panels.
*For a list of assays please go to the nanoString home page and select the “Products” tab.
Sample submission requirements
The assay being used and sample types determine the minimum volume and concentration needed. Please see the nanoString nCounter Analysis sample requirements guide for more information. If you do not have the required sample amount, please contact the ATGC.
Getting started
*First time service users are welcome to request a no-charge consultation meeting. To request a meeting please contact Denaha (D.J.) Doss or Erika Thompson.
- Select and order your assay from nanoString. Please ship to D.J. Doss at MD Anderson Cancer Center, 1515 Holcombe, Rm S15.8425, Houston, TX 77054. Please update D.J. Doss of any shipments expected to arrive at the ATGC.
- There are specific requirements for sample input, depending on the sample type. Please see the sample requirements guide, or contact D.J.Doss for more information about sample quantities.
- The kits are configured for 12 samples so we request samples be submitted in multiples of 12. We can run fewer samples, but the minimum charge is for 12.
- Submit samples along with a completed service request form to D.J.Doss in the ATGC located on the 15th floor of the BSRB, room S15.8425.
- The samples will be checked for concentration using fluorometry (Qubit). An additional QC to check for degradation (Agilent TapeStation) will be performed. The customer will be notified if any samples do not pass the QC.
- Projects are run in the order they are received. If there are no other projects in the queue the data may be available as soon as 4 working days after submission. Runs cannot be started on Fridays.
- Data is presented as raw counts. The investigator will be responsible for analysis using nanoString’s nSolver software. This software is free for download from the nanoString website. Please note: you will need to create an account in order to download the software. The software is also included on the flash drive that accompanies the kit.
ATGC contact information
D.J. Doss
djdoss@mdanderson.org
David Pollock
dpollock@mdanderson.org
Erika Thompson
ejthomps@mdanderson.org
NanoString support contact information
Christof Straub, PhD
Key Account Manager-Houston,Dallas
Email: cstraub@nanostring.com
Christopher DeHeer
Field Application Specialist
Email: cdeheer@nanostring.com
Technical Support
Email: support@nanostring.com
Bionano Optical Genome Mapping (OGM)
What does the platform do?
Visually assess the whole genome by arraying large DNA fragments aka, "Optimal Mapping”, through a non-sequencing based technology allowing a high resolution, SV profiling (SVP).
Most large genomes contain thousands of large structural variants (SVs), repetitive regions composed of identical or similar stretches of sequences, mobile elements such as transposons, large insertions, deletions, translocations, and inversions up to millions of bases, with even partial or entire chromosomes altered.
Some SVs change the dosage of DNA, such as deletions and duplications and are also considered as copy number variations (CNVs) while others, such as inversions and balanced translocation, do not change the DNA dosage. The gain and losses of important genes and regulatory elements due to SVs will impact phenotype causing disease such as cancer and sex development disorders. The SVs caused by the reorganization of the DNA contents connect two distal fragments together leading to gene fusions and chimeric proteins when two distant genes are joined into one. Gene fusions are often major cancer driving events, especially in pediatric cancers and liquid tumors (1).
An extremely complex form of SVs called chromothripsis, in which dozens to hundreds of breakpoints on one or more chromosomes are involved, was originally reported in different types of cancers as well as in germlines genomes causing developmental and neuronal disorders (1).
Bionano Optical Genome Mapping directly observes structural variations by linearizing
and imaging DNA in its native state using massively parallel Nano-Channels.
This direct observation results in some of the longest read lengths in genomic research. As a result, Bionano mapping yields hundreds of times more contiguous assembly than sequencing technologies alone can provide with unparalleled sensitivity for large structural variations (SVs) from 500 bp to mega base pair lengths.
Bionano OGM Applications
- Detects all types of Structural Variants (SVs) down to 5% Variant Allele Fraction for mosaic samples or heterogeneous cancer samples.
- Detects repeats and complex rearrangements.
- Detects genome wide CNVs and fusions, including fusion partners.
- Identify genes of interest, their locations, and how SVs impact them for downstream Applications (2).
Bionano Optical Genome Mapping Technology
Megabase size molecules of genomic DNA are isolated and labelled at a specific 6 or 7 base pair sequence motifs. The motifs occur approximately 8–28 times per 100 kbp, depending on its frequency in a particular genome. The label patterns allow each long molecule to be uniquely identified and aligned.
Labelled DNA is loaded onto a Saphyr chip and placed into the Saphyr instrument where electrophoresis initiates to move mega base length molecules from bulk solution into the silicon chip micro-environment before unwinding and linearizing the DNA in the Nano-Channel arrays. Cycles of loading of the Nano Channels followed by imaging are performed until sufficient data is collected.
Bionano image detection software extracts molecules from raw image data. The backbone stain signal of the DNA molecules is used to identify molecules and to determine their position and size. The distance between the labels on each molecule is recorded to generate an extracted molecule file called a BNX file. The BNX file is the only input needed for the Bionano Results Analysis.
SV calls are made based on analyses of a multiple local alignment between consensus maps and the reference. The pipeline supports calling of major SV types: insertions, deletions, inversions, and translocation breakpoints.
Note: Optical Genome Mapping can detect variation throughout most of the genome however it does not include coverage of centromeres, short arms of acrocentric chromosomes and some exceptionally long paracentric low copy repeat regions.
Bionano Optical Genome Mapping Data Analysis
Bionano Access software enables users to perform a variety of bioinformatic analysis and visualize structural variations (SV) that have been detected through Optical Genome Mapping (OGM) including insertions, deletions, duplications, inversions, translocations, ring chromosomes, complex rearrangements, absence of heterozygosity (AOH) and triploidy.
Analysis Pipelines
- Rare Variant Analysis Pipeline detects SVs genome-wide without bias, including analysis of heterogeneous tumor/mosaic samples down to an average level of detection of 5% variant allele fraction
- De novo Assembly Pipeline calls heterozygous structural variants with unmatched sensitivity and precision
- Copy Number Variation Pipeline detects copy number changes from 500 kbp up to aneuploidies, down to 10% variant allele fraction with high sensitivity
- Variant Annotation Pipeline calculates all SV calls based on the frequency of variants in a built-in control database, and external databases. It annotates calls by providing overlapping gene information, and performs trio-analysis and tumor-normal comparison
Data Visualization: The Circos Plot provides a whole genome summary of variants detected (2).
Data Visualization: The Genome Browser displays alignment of all assembled maps on single Chromosomes (2).
Bionano Optical Genome Mapping Service Requirements
When preparing samples for analysis by Bionano Genome Mapping (OGM) Systems, maintaining the integrity of the samples is critical, as DNA molecules must be 150 kb or larger to be assessed.
Service Scheduling
The OGM Service is by appointment only. Please contact Marisela Mendoza mgmendoz@mdanderson.org to schedule the date and time of sample submission.
Sample Requirements
- Dissociate and harvest cells according to the user’s established protocol.
- We require 1.5 million live mammalian cells from adherent or suspension cell cultures.
- Cells should be submitted in 5ml cold PBS without Calcium or Magnesium.
Note: Samples delivered to the OGM Service by 10 am on the scheduled service date will begin same day processing.
Samples received after 10 am will be prepared for freeze-storage by the OGM Service personnel and placed in queue for processing on the next available date.
Selecting Coverage
Please see the “Initial Structural Variant Analysis” information to select the Coverage and Analysis Pipeline according to the project requirements.
Initial Structural Variant Analysis Performed by the ATGC
| Application | Germline DNA Analysis |
Cancer Analysis |
| Data Collected |
400 Gbp |
1.5 Tbp |
| Coverage Setting |
100X |
400X |
| Effective Coverage |
80X | 300X |
| Variant Allele Frequency (VAF) |
50% | ≥5% |
| Analysis Pipeline |
DeNovo Assembly | Rare Variant Analysis |
Service Output
At project completion, investigators will be provided with access to raw molecules data, assemblies and SV calls as well as a username and password for the Access software to allow for additional analysis.
Note: The ATGC does not provide biostatistical analysis as a service.
Contact Information
Marisela Mendoza: mgmendoz@mdanderson.org
Erika Thompson: ejthomps@mdanderson.org
Viju Varghese: vivarghes@mdanderson.org
References
- Yang, L. A practical guide for structural variation detection in human genome. Curr Protoc Hum Genet. 2020: 107(1): e103
- Visualizing Different Classes of Structural Variants in Bionano Access Software. Bionano Genomics, 2022: Doc 30548, Rev A.