These are image files that can only be opened in the Feature Extraction Software. The jpg image can be opened in a variety of softwares.

For Duke University and other domestic institutions, orders will be invoiced after they have been completed and the data has been released. Pending or uncompleted orders cannot be invoiced. For clients outside the US, orders will be invoiced after they have been completed and data will be released after payment has been received. For big sequencing project ...

For more information about the RNA Ladder we are using, click 

Media Folder: 
PDF icon here
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For total RNA:

RNA quality: For microarray analysis the RIN score should be greater than 7. The ratio for rRNA 28s/18s is also an indication of RNA quality. The ideal ratio is 2, a low ratio is an indication of degradation of the total RNA. On the gel image and the graph, the 28s and 18s should show up as 2 sharp bands/peaks.

RNA concentration: The Agilent RNA quality check can give you an estimate of the RNA concentration, however, we have found it is not as accurate as the NanoDrop measurement. If concentration if what you're concerned about, you are welcome to double-check it on our NanoDrop spectrophotometer. Click here (DEAD LINK) for more information.

For mRNA:

rRNA contamination: the percentage of rRNA in your sample.The ideal number is <5.

RNA concentration: The Agilent RNA quality check can give you an estimate of the RNA concentration, however, we have found it is not as accurate as the NanoDrop measurement. If concentration is what you're concerned about, you are welcome to double-check it on our NanoDrop spectrophotometer. Click here (DEAD LINK) for more information.

If you choose to do your own analysis, we can help you with any supported software listed on out Data Analysis Page. (NEED LINK)

We can also perform data analysis for you.  Click here (LINK) for more information.

The shipping address for NGS samples is

Att:   [insert the name of the sequencing platform you have requested] GCB Genome Sequencing Shared Resource
Rm 119 Biology Bldg
Duke University
130 Science Dr
Durham, NC, 27708
USA

 

Duke investigators must purchase arrays directly from Affymetrix and bring them to the facility when submitting samples. Based on the volume of use, Duke investigators receive a considerable discount on the price of Affymetrix GeneChip® arrays. For more information on array pricing, or to determine the costs for probe synthesis, hybridization, and analysis of Affymetrix arrays, please contact us.

ProjectIDNumber_SubmissionIDNumber_SlideBarcode_ScanNumber_FeatureExtractionProtocol_SlideNumber_ArrayPosition.extension

Example:

1234_9999_11111120110505_S01_HSH-CGH-v4-May05-1_1_1.txt

NanoChip: total RNA: 5-500 ng/µl; mRNA: 25-250 ng/µl

PicoChip: total RNA: 0.2-5 ng/µl; mRNA: 0.5-5 ng/µl

The Affymetrix GeneChip® system provides an approach to comparatively analyze genome wide patterns of gene expression using a technology that incorporates miniaturized, high density arrays of 25mer oligonucleotide probes. The probe arrays are manufactured by Affymetrix's proprietary, light directed chemical synthesis process, which generates high density arrays of oligonucleotides that possess a predefined position on the array. These arrays are used to monitor gene expression for thousands of transcripts.

There are several types of Affymetrix GeneChip® arrays. Our facility primarily works with three kinds: 3' expression arrays, Whole Transcript (Exon and Gene) arrays, and miRNA arrays.

1. 3' Expression arrays: these arrays have been a microarray platform standard for many years, with large quantities of published data available for comparison, and well developed functional annotations of the genes represented. Transcripts are represented as probe sets, where a probe set is made up of probe pairs comprised of perfect match (PM) and mismatch (MM) probe cells. This probe pairing strategy identifes and minimizes the effects of non specific hybridization and background signal. The intensities of each probe pair are used to determine the expression measurement. This measurement is calculated for each probe set and is described in the form of qualitative and quantitative values using the Affymetrix GeneChip® Command Console software, v.1.1. Array quality control metrics are well developed and understood for these arrays.

Target preparation from totalRNA uses T7 primers (incurring a 3' bias of transcript sensitivity) in the cDNA synthesis step, followed by in vitro transcription to produce biotin-labeled cRNA. The cRNA is fragmented and mixed into a hybridization cocktail before application to the array, which is then hybridized for 16 hours at 45C with rotation at 60rpm.

2. Whole Transcript (WT) arrays: these arrays have been developed more recently to enable analysis of the entire length of the genomic transcript, not merely the 3' end. This offers the advantage of more complete and precise expression information, and may provide new insights. However, there is not yet a wealth of published data avaialble for comparison and statistical analysis methods have not yet been standardized. Very little array quality control metrics are available.

  • Exon 1.0 ST arrays offer the most comprehensive analysis, with both alternative splicing information (exon-level analysis) and gene expression information that is derived from multiple probes on different exons and summarized into a single value for all transcripts from the same gene (gene-level analysis).
  • Gene 1.0 ST arrays are available for human, mouse, and rate, and offer a reduced WT approach that enables gene-level analysis for the well-annotated genes at a more affordable cost than the Exon 1.0 ST arrays.

Target preparation from totalRNA uses T7 primers in the cDNA synthesis step, followed by in vitro transcription to produce cRNA. Random primers are then used to synthesize cDNA from the cRNA. The cDNA is fragmented, biotin-labeled, and mixed into a hybriziation cocktail before application to the array, which is then hybrized for 17 hours at 45C with rotation at 60rpm.

3. miRNA arrays: released in 2009, these arrays provide expression analysis pioneers a tool to detect non-coding RNAs, including human, mouse, rat, canine, and monkey miRNAs, Sanger miRNA database V1.1 content and additional human small nucleolar RNAs (snoRNAs and scaRNAs). Very little data has been published, and few array quality control metrics are available.

Target preparation from totalRNA uses Genesphere® FlashTag Biotin Labeling kit. After poly-A tailing, the totalRNA is biotin-labeled in the FlashTag Ligation step, and mixed into a hybridization cocktail before application to the array. The array is hybridized for 16 hours at 48C with rotation at 60rpm.

Immediately following hybridization, the hybridized probe arrays undergo an automated washing and staining protocol on the GeneChip® FS450 fluidic station and are then scanned on the GeneChip® Scanner 3000 7G Plus where patterns of hybridization are detected. The hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the target, which is now bound to the probe array. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Data generated from the scan is then analyzed using the Affymetrix GeneChip® Command Console software, v1.1, and the Affymetrix GeneChip® Expression Console software, v1.1. Affymetrix miRNA data is analysed using the miRNA QC Tool.

Feature Extraction is the process by which data is extracted from the scanned microarray image (.tif) and translated into log ratios, allowing researchers to measure DNA copy number changes in their experiments.

Agilent MicroArray Design Identifier

The first 6 digits of the design file indicate the AMADID. For example, if the design file is 012097_D_20070820, 012097 is AMADID and 20070820 is YYYYMMDD that the design file was generated.

Have I said thank you recently? You and Darren are being so helpful to all of us.

 RNA samples must be resuspended in  nuclease-free water. Samples should be submitted in an eppendorf tube clearly marked with your initials and a 2 to 3 digit identifier as well as your order number. See input amount for the various NGS platforms at xxxxx

For each array experiment that is performed using Agilent arrays, four files with identical names but different extensions will be generated (*MAGEML xml, *txt, *pdf, *jpg).

For each array experiment that performed using the Affymetrix GeneChip® arrays, six files are generated (*.DAT, *.CEL, *.ARR, *RPT, *.txt).  Each file will be named identically except for the extension.  The naming convention is as follows:

Project ID number_Submission ID number_Genome ID number_samle name_chip type, i.e.  1234_0001_12123_CX24R_HU133+2.*

In the case of pairwise comparisons in the Affymetrix Microarray Suite v5.0, the comparison files will follow the following naming convention: Sample_base_Sample_exp.txt

All data analyses will be given as *.txt files.

The types of extensions are as follows:

ExtensionDescriptionComments
*.DATScanned image of the GeneChip arrayCan only be opened in Affymetrix
*.CELCell intensity file that calculates the average intensities for each cell and assigns it to an x,y cooridinate posiionCan only be opened in Affymetrix GeneChip Expression Console
*.CHPContains analysis outputCan be opened in Excel to manipulate
*.ARRContains experimental informationCan only be opened in Affymetrix
GeneChip Expression Console
*.RPTContains quality control information about the chipCan be opened in Excel to manipulate
*.txtContains analysis outputCan be opened in Excel to manipulate

DNA samples must be resuspended in  nuclease-free water. Samples should be submitted in an eppendorf tube clearly marked with your initials and a 2 to 3 digit identifier as well as your order number. See input amount for the various NGS platforms at xxxxxx
 

Ensure that your DNA sample:

  • Is double-stranded. Single-stranded DNA will not be made into a sequencing library and can interfere with quantitation and polymerase binding.
  •  Has undergone a minimum of freeze-thaw cycles.
  • Has not been exposed to high T (>65°C for 1 h can cause a detectable decrease in sequence quality).
  • Has not been exposed to pH extremes (<6 or >9).
  • Does not contain insoluble material, and is not colored or cloudy.
  • Does not contain RNA.
  • Has not been exposed to intercalating fluorescent dyes or UV radiation.
  •  Does not contain chelating agents (e.g., EDTA), divalent metal cations (like Mg 2+), denaturants (like guanidinium salts, phenol), or detergents (like SDS, Triton-X100).
  •  Does not contain carryover contamination from the starting organism/tissue (like heme, humic acid, polyphenols).

    The PacBio template preparation process does not utilize amplification techniques; therefore, the input DNA quality will be directly reflected in the sequencing results. Any DNA damage (e.g., basic sites, nicks, interstrand crosslinks) or contaminants (e.g., single-stranded DNA, RNA, proteins, dyes, or salts, phenol) present in the input material will impair performance of the system.

Go to the Affymetrix website to view the various GeneChips® available. Go to "Tools & Data" under each chip to download genelists.

Yes you may submit your own library(s) for Illumina sequencing. The concentration of your library should be >10nM in more than 15μl; EB (Tris-Cl 10mM, pH 8.5).
    We will perform QC on your library by Qubit and Bioanalyzer or Tapestation, and inform you if your library does not meet our expectations.
    You must supply all index information for your libraries when you submit your samples.  Please email the completed index template (link in your DUGSIM order) to sequencing@duke.edu.
    We do not guarantee the sequencing results from user prepared libraries.
 

You will be notified by email after your sequencing run has completed, and the data has passed our QC.  The notification email will also contain instructions for data retrieval.

  • MiSeq data is released through Basespace and will be demultiplexed FASTQ files.
  • HiSeq  data will be available as FASTQ files.  The data will be demultiplexed. All reads are raw reads.
  • PacBio data is released as raw read data (HS, Fasta) along with FASTQ subread files.

 Please note that your data will only be available for one month after it is released.

After you have received your order number, samples should be shipped on  dry ice in  1.5ml tubes. Tightly closed tubes should be well labeled with the sample name (your initials followed by a number). Please write the sample name on both the side and the lid of the tube. Please also write your order number on the side of the tube. If you use a sticker  for a sample label, make sure it is freezer compatible. We recommend stickers from GA international (www.Labtag.com) , LT-11. Place your sample tubes inside a small cardboard box, a large tube or bubble wrap so that the tubes will not be broken by the dry ice  during transit. Please include a printed copy of your order from DUGSIM and a completed sample submission sheet. If you are submitting prepared libraries, please email a completed index template to sequencing@duke.edu.  (link to template is on your order in DUGSIM). We cannot guarantee the fate of any samples received without the above requested documentation.
 

Please give us 3 µl of each RNA sample in 0.5 tubes. 1µl is run on the Agilent Bioanalyzer (but we might need to rerun the sample in case it didn't run well the first time). For quality check prior to microarray analysis, 1 µl is also needed to measure concentration and absorbance ratios on NanoDrop.

Only the sample of interest is necessary (a reference sample is not used).  Samle guideliens are as follows:

  1. 3' Expression Arrays: for complete processing, at least 200ng** of totalRNA in 8μl RNase-free water; for hybridization only from Affymetrix protocols, 20μg fragmented biotin-cRNA in 40μl volume (49 Format array) OR 15μg fragmented biotin-cRNA in 30μl volume (100 or 169 Format array) is required, according to the protocol; for hybridization only from NuGen WT-Ovation Pico protocol, 5μg fragmented and labeled cDNA in 50μl volume, according to the protocol.  **Note: At least 2μg in 8μl RNase-free water is required for the "old" Affymetrix One-Cycle IVT processing.
  2. WT Arrays: Exon-for complete processing, at least 1μg of totalRNA in 8μl RNase-free water; for hybridization only, 5.5μg fragmented and labeled ssDNA in 60ul volume, according to the protocol.  Gene-fore complete processing, at least 100ng of totalRNA in 8μl RNase-free water; for hybridization only, 5.5μg fragmented and labeled ssDNA in 60ul volume, according to the protocol.
  3. miRNA Arrays: for complete processing, at least 1μg of totalRNA in 8μl RNase-free water.

For procedures starting from totalRNA we also perform an RNA quality check, so please bring an extra 3μl aliquot of your samle in a seperate tube.

We have a online system named DUGSIM (Duke University Genome Sequencing Information Manager) that allows you to request a formal quote and place an order: https://dugsim.net/

After reviewing the cost estimate and/or consulting with us, if you are interested in placing an order, you will need to Sign up in DUGSIM (link on page explaining on to do that).
Duke clients are already pre-approved for directly placing an order for Sanger Sequencing and Fragment Analysis. However, for next-generation sequencing, all clients must first request a quote.

After approval of the quote by us, you will be able to place an order for available services on any of our sequencing platforms.
Note that because of our tax-exemption status, we are not allowed to provide services to for-profit organizations.

A few options are available to you.

1)  you can go to https://dugsim.net/estimate_cost 

2) You can contact us directly by email at sequencing@duke.edu

3) You can create an account on DUGSIM  https://dugsim.net/sign_up  and go to the "Estimate Cost" page


 

Sign in to your DUGSIM account at https://dugsim.net/sign_in

From the main page, select “Invoices” from the top menu bar.

Find the order for which you need an invoice, and select the “Details” link for that order.


On the resulting display, scroll to the bottom of the order and select the “Printable” button. This will take you to a printer-friendly display of the order.
From that page, you can print or convert to pdf using your browser’s print function.

 Yes, as long as you submit those custom sequencing primers along with your libraries.  We will also require you send us the  complete information about your primers (sequence and concentration). We can only accept custom sequecing primers for orders on the MiSeq and HiSeq Rapid Run.
Custom Sequencing Primers should be >15 ul. at 100uM.

 

 

 

We will pool up to 12 libraries for your sequencing order.
Additional numbers of libraries will incur an extra fee.