Sequencing Facility

Microarray Facility

Guidelines for Sample Submission

Please contact the Sequencing Facility Manager when sending samples. There are two options for sending samples:

Schedule an appointment to bring samples to the facility.
Email the facility manager
Mindie Lipphardt or call 304 293-5201 ext 31480 or ext 31512

or

Ordering Sequencing or Genotyping Service

This Genomics Core Facility website allows you to upload a file (comma or TAB-delimited text file) that specifies sequencing information. Additionally, you can also upload a Gel image. Below you'll find information about proper formatting and example files. Ordering and sample information uploads can be accomplished on the Sequencing Orders page

Proper File Format

The purpose of the flat-files is to specify well information for your sample sequencing. The genetic sequencer uses a plate that contains 96-wells. Of those 96 available wells, your flat-file must specify which wells to use, their contents and parameters. You can specify up to 96-wells per work order and thus per flat-file. You cannot use multiple flat-files for one order. If you need more than 96 wells, please submit multiple orders to do so.

Working with Wells

Each well on the plate has a specific alpha-numeric value associated with it. There are eight alpha-columns (A-H) and twelve numeric-rows (1-12) per plate for a total of 96 wells (A01 through H12). The specification is alpha-column first and numeric-row second, like A05. The system allows you to specify which well to begin on but MUST be an ODD numeric-row and must be an A like A11.In order to start on a specific well, you must upload a flat-file. You cannot manually enter samples and start on arbitrary well. In all other cases, the system assumes you're starting on well A01.

You can optionally omit the Well information from the flat-file. The system then assumes that you are starting on well A01. You can additionally specify only the initial starting well and omit the others, the system will increment the remaining wells for you. If you specify an arbitrary starting well, you must ensure you stay within the 96-total wells of the plate, otherwise the flat-file will be rejected. Again, if you wish to specify arbitrary starting well it must be one of the possible Axx wells, xx being an odd number.

Flat-File Format

The system expects the sequencing well information in lines and ignores blank lines. It also expects well-specific information in columns as outlined below. The columns can be separated by commas or by TABs.

Here are the default required columns:

Well

Template Name

Template Concentration (ng/uL)

Name of Primer

Primer Concentration (pmole/uL)

Vector Name

Size of Insert (bp)

The column names above in general should be specified in the flat-file upload but could be entirely omitted if you choose. If you include the column names, you must make it the first non-blank line of the file and you must spell at least the first two columns exactly as shown above (exluding the units in brackets) in order for the system to properly recognize them. That is, you must specify at least the first two columns for it to recognize the line as the column header. Therefore, if you name your actual well data like the column header on the first non-blank line, the system will treat it as the column header. Moreover, whether you specify the columns or not, the order in which the columns appear above, from left-to-right, are the order the system expects for your data. That is to say, you cannot rearrange the column header and expect the system to accept it.

Note that the system ignores blank lines but DOES NOT ignore extra TABs in-between columns. That is, if there are multiple TABs between column names or data, it will count each TAB and possibly cause the upload to fail.

Omitting Information

As mentioned, the system is rather dynamic in that you can omit various pieces of information and the upload will still work. For example, you can sometimes omit the Wells column if you wish, the system will assume you are starting with well A01.

You can specify only the Wells and Template Name when running Option C from the Order and Services page. If you run Option A or B, however, you must specify the all the column information in the flat-file, even if you upload a Gel image (with the exception of omitting alpha-numeric Well data). If you specify a Gel image and run Option A or B, you can fill-in fake data as placeholders. We recommend using the examples below as your starting point since they already contain fake/dummy data.

When specifying the Template Name in any case, you MUST specifty unique names for each well. The only exception is when specifying a blank well as described below (that is, you can have multiple Template Names with "blank" specified).

Blank Wells

You can specify blank wells (in-between actual, used wells) if you wish. In order to do so, specify Blank or F for the Template Name in the file. Also note that the specification is not case-sensitive, so blank or f would work also. If you wish to specify blank wells, you must upload a flat-file. Blank wells before or after the used wells in the plate are taken care of for you.

Example Flat Files

Any of the following samples will work with Option C (both Fragment and Non-Fragment Analysis) in the Orders and Services section. However, only the examples that contain all columns (Temp. Name, Temp. Conc., Name of Primer, etc ) will work with Options A and B.

All Features Explicitly Included

All Features Without Column Header

Column Header and All Columns Except Wells

Column Header With Wells and Template Name Only

Template Name Only

Microarray Core Facility

Guidelines for sample submission:

Please contact Microarray Facility when planning GeneChip experiment to ensure the availability of the number of chips to be used. It usually takes one to two weeks to order and receive the chips.

Schedule an appointment to bring samples to the facility.
Email or call Viola
Wszeszel-fedorowicz@hsc.wvu.edu
Tel. # 304-598-4744

Completion of Sample Submission Form is required at the time of sample submission. Sample names must be clearly labeled on top of the tube and must match names on the request form or they will be returned. Sample names cannot be longer than 12 characters in length. All RNA samples must be DNase treated prior to submission to ensure all contaminating genomic DNA has been removed. We recommend that you resuspend or elute your RNA in DEPC water.

If you are submitting samples for nucleic acid quantification via the Nanodrop you must submit an aliquot of the elution buffer with your samples. For Nanodrop quantification please submit 3 ul of your sample.

The quality of total RNA has to be check via 2100Bioanalyzer (Agilent Technology). 2 ul of sample should be submitted for Bioanalazer analysis.

Investigators are responsible for the cost of RNA concentration and quality assessment performed in Genomics Core Facility, unless they can provide Nanodrop and Bioanalyzer files / printouts at the time of sample submission.

The total RNA, provided to the facility should be at the minimum 2ug, in a concentration no less than 0.31ug/ul in DEPC-H2O. With the smaller amount of total RNA amplification method is required, which can be done at Genomics Core.

For additional services such as RNA isolation, please contact Genomic Core Facility.

After microarray assay a DVD will be provided to the investigator including the .dat, .cel, and .chp. Absolute text files or excel files can be generated upon request. The Genomic facility will archive a copy, but we suggest that you also make your own back-up. The facility will generate a report summarizing all steps, quality control checks, and results for potential troubleshooting.

RNA isolation:

The quality of the RNA is important to the overall success of the microarray analysis. Since the most appropriate protocol for the isolation of RNA can be tissue-dependent, we recommend using a protocol that has been already established in the laboratory. In the absence of an established protocol, Affymetrix suggests using one of the commercially available kits designed for RNA isolation. If mammalian tissue is used as the source of RNA, Affymetrix recommends isolating total RNA with a commercial reagent such as TRIzol.

Isolated RNA should be dissolved in RNase-, DNase-free H2O. The appropriate volume for resuspension depends on the expected yield and the amount of RNA required for the cDNA synthesis.

Quantity of RNA should be determined by spectrophotometric analysis using eg. using a Nanodrop. which can be done by the Genomics Core facility personnel. The absorbance should be checked at 260 and 280 nm for determination of sample concentration and purity.
1 OD at 260 nm equals 40 ug per ml.
The A260/A280 ratio should be close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable).

Quality of RNA should be checked using the Agilent 2100 Bionalyzer, which can be done by the Genomics Core Facility personnel.
Note: Although the Agilent Bioanalyzer gives a concentration, the NanoDrop gives a more accurate reading.

Director: Stephen DiFazio. (stephen.difazio@mail.wvu.edu)
Lab Manager: Alex Harris (wvugenomics@gmail.com)