The Sequencing and Microarray Facility (SMF) offers a variety of services, including:
Includes next-generation sequencing, Sanger-based DNA sequencing and gene resequencing and single cell mRNA-Seq
The SMF offers several additional services, including:
- Droplet digital PCR
- Fluorescent fragment analysis
- nanoString nCounter analysis
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
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 SMF’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 SMF.
- The investigator sets up the Eva Green or TaqMan-style, probe-based reactions in a 96-well plate (Bio-Rad P/N 1864108 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 1864108).
- The investigator delivers the reactions in a sealed 96-well plate to the SMF’s ddPCR service.
- The SMF 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.
- Complete the Service Request and Plate Layout forms and email them to: ddPCRSubmissions@mdanderson.org. Contact the SMF for the forms.
- Provide the reactions in a 96-well plate (Bio-Rad P/N 1864108 preferred), sealed and centrifuged.
- Results can be expected the same day, provided the plate is submitted before noon.
SMF contact information
Denaha (D.J.) Doss
Bio-Rad support for assay design or data analysis
By telephone: 1-800-4-BIORAD (1-800-424-6723)
By email: firstname.lastname@example.org
Fluorescent Fragment Length Analysis is used to determine genotypes and assess loss of heterozygosity and microsatellite instability. 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.
View the service pricing schedule for more information about fluorescent fragment analysis pricing.
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 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. The addition of the sequence tail GTTTCTT on the 5' end of the reverse primer is one way to force the "+A" reaction to completion (Browstein et al.,1996). 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 recommends using 5' end-labeled primers. Please label only ONE of your primers.
- Size standard: We use an internal standard that ranges from 35-500 bp. As a result, we strongly recommend that your PCR products be between 50-500 bp in size. The internal standard that our software uses to analyze the fragment lengths is labeled with ROX (red) so we do not recommend using this label for your primers.
- 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 an 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 0.5-2 ng/µl.
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 hundred 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).
The nCounter MAX/FLEX system consists of a robotic nCounter Prep Station for sample processing and the nCounter Digital Analyzer for collecting data.
NanoString offers a variety of pre-designed, off-the-shelf assays*. The SMF stocks some commonly requested assays (please ask us about our in-stock 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 SMF.
*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 an in-stock assay or order your assay from nanoString. Please check with D.J. Doss for information about which kits are stocked at the SMF.
- 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 SMF located on the 15th floor of the BSRB, room S15.8425.
- The samples will be checked for concentration using fluorometry (Qubit) and the quality is checked with a NanoDrop. An additional QC to check for degradation (Fragment Analyzer) 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.
SMF contact information
NanoString support contact information
Field Application Specialist