Platform:
Fragment Analysis
Category:
Microsatellite Genotyping-Human Cell Line Validation-CODIS
Brief Description:
The Fragment Analysis service offers the AMPFLSTR Identifiler Plus Assay for cell line identification sold by Applied Biosystems. This assay does identify Human Genomic DNA for 15 tetranucleotide repeat loci and the Amelogenin gender determination marker. Our 3730XL Genetic Analyzer uses five fluorescent dyes per lane permitting multiplexing of four microsatellites and a fifth dye for an internal size standard to correct for variability between lanes.
Primary Contact:

Suzanne Genik
genik@umich.edu
734-764-8067

Typical Cost:

The cost for each Identifiler Plus sample is $32.00. This includes PCR reactions performed by the core, samples run on the 3730XL DNA Genetic Analyzer and analysis of data with GeneMapper software. The cost for processing of Identifiler Plus samples submitted from outside the University is $41.28 per sample.

Typical Output:

Raw data .fsa files are generated for each sample. GeneMapper software is used to analyze the data and .pdf documents are generated of the analyzed data. HID GeneMarker software from SoftGenetics is available for clients to check out and analyze data themselves.

How to request:

Sample Submission: go to

https://client-seqcore.brcf.med.umich.edu/ ​ then choose ‘Fragment Analysis Service, Submit Samples for IdentifilerPlus’.

Sample requirements:

Submit 20ul of DNA sample at a concentration of 0.1 ng/ul in Ultra pure RNase, DNase free water or EB Buffer.

 

Typical Turnaround:

Between 1 to 3 days. The Identifiler Plus samples take a couple of days to process. 

Critical information:

Submit samples in 1.5 ml microcentrifuge tubes. It is important to Label each tubes with the identification number assigned by our database on submission of your samples.

More information:

The HID GeneMarker software is available to check out at room 2568 MSRB II.

Clients must have an account set up with the DNA Sequencing Core prior to submission of samples. The clients can use any of our drop off locations listed on our web page: http://seqcore.brcf.med.umich.edu/.

Identifiler Plus samples received from outside the University of Michigan can be shipped to: University of Michigan, DNA Sequencing Core, NCRC Bld 14, Room 122, 2800 Plymouth Road, Ann Arbor, MI 48109-2800.

Platform:
PyroMark Q96 MD and PyroMark Q96 Vacuum Workstation
Category:
Genotyping
Primary Contact:

Ellen Pedersen

Research Specialist: Sequenom and PyroMark Services

elleped@umich.edu

University of Michigan DNA Sequencing Core - NCRC Building 14 Room 134

734-615-5646

Typical Cost:
Service Cost

96-well plate

$320

 

Typical Output:

Plate Report (text or Excel file) containing allele quantification percentages and sample/well quality metrics, along with Pyrograms depicting spectra for each well/sample.

How to request:

Contact Dr. Lyons for initial consultation; if he deems project appropriate for PyroMark, contact Ellen Pedersen.

Sample requirements:

Sample prep according to Qiagen’s specifications; Ellen will arrange direct support from Qiagen to ensure optimization of PCR products prior to sample submission for pyrosequencing.

Typical Turnaround:

Depends on project queue; each plate/run requires ~4 hours to process.

More information:

If Assay Design is completed by Core, the costs will involve additional labor charges billed at the current aprroved PyroMark Tech time hourly rate, which will be added to the current per plate recharge rate.

Platform:
Pacific Biosciences PacBio RSII
Category:
Single Molecule Real-Time (SMRT) Sequencing
Primary Contact:

Christina McHenry, PacBio Sequencing Specialist (cmchenry@umich.edu)

Typical Cost:
  • Covaris gTube shearing for long template sequencing per sample = $25.00
  • Template library production per sample = $250.00
  • PacBio sequencing per SMRT cell = $190.00
  • Additional for external customers = add 29%

Libraries vary widely in yield and therefore the number of cells that can be run. Repeat libraries from the same sample tend to give similar yields.

Rough estimate is 200 Mbases of data per SMRT cell (i.e. 100x coverage of a 4 Mb genome for De Novo sequencing would be estimated to require 2 SMRT cells.)

Typical Output:

Current results are approximately 40,000 to 90,000 reads at 3,000 to 15,000 bp average read length, depending on type and quality of sample and the specific sequencing method requested. Longest individual reads may reach 70,000 bp. Output can vary greatly for different samples due to type of sample (e.g. short templates run better than long ones) and individual characteristics of the DNA (damage or contaminants that inhibit the polymerase, etc.) but we generally assume that a good sample will yield 40,000 sequences at 10 kb average read length, for a total of 400 Mb of data output. Your result will probably be different!

How to request:

Sample Submission: Please see the 'Submit Samples' menu, above.

Sample requirements:

Amounts and concentrations of sample vary depending on the type of project and desired insert size for the templates. General ranges:

  • 250bp or 500bp without shearing = 250 ng dsDNA in 23 ul or less
  • 1 Kbp or 2 Kbp without shearing = 500 ng dsDNA in 23 ul or less
  • 5 Kbp without shearing = 2000 ng dsDNA in 39 ul or less

(Double the required amount above if shearing is needed.)

Genomic or other large DNA that requires shearing to 10-20 Kbp fragments for long template sequencing requires a starting amount of 10,000 ng dsDNA in 300 ul or less for two rounds of 150 ul shearing in Covaris gTubes.

Ask us if your sample does not easily fit into one of these categories. We can usually figure an amount that will work. Keep in mind that the smallest fragments in a mixture will tend to sequence preferentially so a mixture is best if the sizes are similar or if the bias for smaller sizes is acceptable.

Sample submission recommendations from Pacific Biosciences:

The Pacific Biosciences template preparation process does not utilize amplification techniques. As a result, input DNA quality will be directly reflected in sequencing results. Any DNA damage present in the input material (abasic sites, nicks, interstrand crosslinks, modified bases, etc.) will result in impaired performance in the system.

Therefore, ensure that your DNA sample:

  • Is double-stranded. Single-stranded DNA will not receive end-adapters in this template preparation process and can interfere with quantitation and polymerase binding. Qubit fluorometer or PicoGreen assay are both good for determining double-stranded DNA concentration. If you are quantitating with a Nanodrop or other spectrophotometer, please send excess as they tend to read other components besides dsDNA and overestimate the concentration.
  • Has undergone a minimum of freeze-thaw cycles.
  • Has not been exposed to high temperatures (65ºC for 1 hour can cause a detectable decrease in sequence quality), pH extremes (< 6 or > 9).
  • Has an OD260/280 ratio of approximately 1.8 to 2.0.
    An OD 260/230 of at least 2.0 is also recommended to avoid carbohydrate, phenol, and other contaminants.
  • Does not contain insoluble material.
  • Does not contain RNA contamination.
  • Has not been exposed to intercalating fluorescent dyes (e.g. ethidium bromide) or ultraviolet radiation (e.g. transilluminator.)
Typical Turnaround:

Usually 3-4 weeks, unless we have received abnormally large numbers of samples submitted, or the sequencer has been down for maintenance or repair.

Critical information:

The sample will not be amplified so the quality of the DNA directly affects the sequencing performance. Concentration is best determined by a Life Technologies Qubit Fluorometer or a PicoGreen Assay, both of which only measure double stranded DNA. If you use a Nanodrop or similar spectrophotometer, please send extra sample as they tend to give higher readings due to detection of other components.

More information:

PacBio templates are produced by ligating single stranded end adapter loops to both ends of a double stranded DNA fragment. This creates a functionally circular template. The DNA polymerase will continue around the circle until the end of the observation period or until the polymerase becomes inactive. This allows short fragments to be read at high accuracy by creating a consensus sequence from multiple passes across the insert. Long fragments that are read only once will have an error rate around 15% but the long length allows scaffolding for contigs. Errors in PacBio sequencing tend to be random and so are easily correctable by generating consensus sequences. PacBio sequencing is tolerant of low complexity regions such as long repeats. Data is returned as FASTA and FASTQ files and metadata files for bioinformatics, with additional CCS (circular consensus sequence) files on request. Due to the unusually long reads and error rate, PacBio data is best analyzed by a Bioinformaticist using Pacific Biosciences SMRT Portal secondary analysis software (available from their website.) See link for information on bioinformatics analysis:

https://github.com/PacificBiosciences/Bioinformatics-Training/wiki

Researchers at the University of Michigan can contact the Director of the Bioinformatics Core to arrange for secondary analysis of PacBio data.

Platform:
Illumina HiSeq Libraries
Category:
Multiplexing
Primary Contact: Typical Cost:
Internal

$19 per sample

External

$24.51 per sample

 

Typical Output:

Balanced pool of sequence ready libraries.

How to request:

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’​​

Sample requirements:

More than one library per sequencing lane.

Typical Turnaround:

2-3 days

Critical information:

To achieve a similar number of reads per sample special care must be taken when pooling samples for sequencing. Samples are carefully quantified, balanced and pooled.

Platform:
Illumina Library prep
Category:
Gel Size Selections
Primary Contact: Typical Cost:
Internal

$24 per sample

External

$30.96 per sample

 

Typical Output:

Size selected sequence ready libraries

How to request:

Add to the notes section “Please gel size select between X and Y bp.”

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’​​

Sample requirements:

Refer to the sample requirements of your requested library prep type.

Typical Turnaround:

2-3 days

Platform:
Illumina HiSeq Libraries
Category:
Genome Sequencing (and gene expression profiling if used in conjunction with SMARTer kit)
Primary Contact: Typical Cost:
Internal

$126 per sample + $19 per sample for multiplexing

External

$162.54 per sample + $24.51 per sample to multiplex

 

Typical Output:

The Rubicon ThruPlex/Clontech Low input library prep kit will yield enough library to sequence on an Illumina HiSeq

How to request:

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’

Sample requirements:

The total fraction of 50pg – 50ng full length, of fragmented double strand DNA.

 

Typical Turnaround:

3 weeks

Critical information:

DNA is sheared by sonication on the Covaris. Sheared DNA is end repaired adapter ligaed and PCR amplified.

Platform:
Illumina HiSeq Libraries
Category:
Genomic DNA Seqeuncing
Primary Contact: Typical Cost:
Internal

$149 per sample + $19 per sample for multiplexing

External

$192.21 per sample + $24.51 per sample to multiplex

 

Typical Output:

Illumina ready library

How to request:

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’

Sample requirements:

1 ug to 3ug of good quality DNA in 25-50 ul

Typical Turnaround:

3 weeks

Platform:
Illumina HiSeq Libraries
Category:
Genome Sequencing
Primary Contact: Typical Cost:
Internal

$69 per sample +$19 per sample for multiplexing

External

$89.01 per sample + $24.51 per sample to multiplex

 

Typical Output:

The Wafergen reagents on the Apollo robot will yield an Illumina ready to sequence library with an insert size between 200-550bp.

How to request: Sample requirements:

Good quality DNA 0.1 to 3ug in 25-50 ul

Typical Turnaround:

3 weeks

Critical information:

DNA is sheared by sonication on the Covaris. Sheared DNA is end repaired adapter ligated and PCR amplified.

Platform:
Illumina HiSeq Sequencing
Category:
Rapid Paired end 150bp Sequencing
Primary Contact: Typical Cost:
Internal

$2670 per lane

External

$3444.3 per lane

 

Typical Output:

40 Gb per lane

How to request:

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’​​

Sample requirements:

30 ul of 10nM Illumina library with an insert size of at least 350 bp

Typical Turnaround:

The run itself takes 40 hours the flow cell needs to be full for us to run it. Each flow cell has two lanes and we need to have a rapid mode HiSeq available. This could take some time to fill and run.

Critical information:

Rapid modes come at a premium while they are much faster run times they produce about 80% of the data as compared to a high output run.

Platform:
Illumina HiSeq Sequencing
Category:
Rapid Paired end 125bp Sequencing
Primary Contact: Typical Cost:
Internal

$2390 per lane

External

$3083.10 per lane

 

Typical Output:

45 Gb per lane

How to request:

Sample Submission: login through https://client-seqcore.brcf.med.umich.edu/ then choose ‘Illumina Sequencing’​​

Sample requirements:

30 ul of 10nM Illumina library with an insert size of at least 350 bp

Typical Turnaround:

The run itself takes 32 hours the flow cell needs to be full for us to run it. Each flow cell has two lanes and we need to have a rapid mode HiSeq available. This could take some time to fill and run.

Critical information:

Rapid modes come at a premium while they are much faster run times they produce about 80% of the data as compared to a high output run.