Nevada Genomics Center

Transcription

1 Nevada Genomics Center These are general instructions on how to use dnatools to submit Sanger sequencing samples to be run on the ABI Prism 3730 DNA analyzer. We here at the Nevada Genomics Center feel that dnatools is user friendly and fairly intuitive; but we still recommend taking a moment to read through these instructions as well as taking time investigate dnatools and read the Learn More options that can be found throughout. Please share this PDF file with the rest of your lab. If you have any difficulties using dnatools, please phone ( ) or us (genomics@cabnr.unr.edu) and we will assist you. How to use dnatools for Sanger Sequencing on the 3730 Table of Contents Creating an Account 2 Entering a Sequencing Request 3 Uploading and Importing a Request 7 Viewing your Orders 9 Deleting an Order or Request 10 Display Order Summary 11 Viewing and Downloading Your Sequencing Results 12 Viewing the Chromat 13 Downloading 15 Creating a Fasta Library 16 Searching Sequencing Results 17 PI Group Results View 18 Job Quality Report 19 1

2 Creating an Account You will find a link to the dnatools server on the NGC homepage at If you do not have an account, choose the Create Login Account for dnalims option and fill out the form with a login name, password, your first and last name, address and phone number. Then select the name of your PI from the drop-down list of PI s and this will auto fill the Billing and PI Information. If your PI does not yet have an account then please NGC to have an account opened for your PI. When creating your login information, do not use the following characters: < > / ; " = Login Name and Passwords must have at least 6 characters. The Passwords must be 6 characters in length, have a numeric character (0-9) AND one of the following special &? : + Step #1 Fill-in Login Info Step #2 Select your PI from drop-down list Step #3 Select Submit at the bottom of the form. Once you have set up your account then you can choose the Login to dnalims to enter the dnatools server. 2

3 Entering a Sequencing Request Under the heading Sequencing you will see options in black (for sequencing samples and a summary of your orders), brown (for fragment analysis samples), or blue (to upload and import either sequencing or fragment analysis). To enter a sequencing request you may use either the Enter DNA Sequencing Requests link or you may Upload and Import. First, we describe the use of Enter DNA Sequencing Requests. Select this link and you will open a page that lists the options available for that service. Enter the number of samples you wish to submit and choose the type of service from the dropdown box. There is a maximum of 95 samples per sequencing job request. If you have more than 95 samples you need to submit more than one job request. There is a description of each service in the table under the dropdown 3

4 box. If you are not sure which service you need contact NGC. Once you have filled in the boxes, click the submit key. The next page to open is a table where you enter payment information, sample and primer names, and information about each sample. The payment options are Purchase Order, Account Name, or credit card. When you select your payment option a dialog box will come up so that you can enter the PO, Acct Name, or credit card. If the dialog box does not appear, check to make sure you do not have pop-ups blocked while working in dnatools. If entering credit card information note that must be hand written on the printed form never type that information into the server. UNR campus users should choose Acct Name and enter the PI s last name. If your UNR lab uses more than one PCard then please also add either the last 4 digits of the PCard number or enter the name of the account that you have established with us in the comments field. Select whether you have already added the primer, or not, and if there is primer in some, but not others you should make a note of that in the comments box. Then you can enter your sample names in the table. Filling in the sample names table: Sample Each request is required to have a sample name. The sample name has specific restrictions: The sample name must be more than one character but cannot longer than 20 characters. Use only legal characters. Legal characters are: a-z, A-Z, 0-9, and - Illegal characters are any other characters. Do not use underscore. There are numerous convenient tools to assist you with filling in sample names. Click the the options are described. and all 4

5 Amount, ng or Unknown Enter the amount of template DNA in each sample. If you are unsure as to how much template to use, please refer to the NGC web page for guidelines. If you are having us quantify the template and set-up the reaction then list the amount as unknown. Volume or Dry Enter the volume (in ul) you that you are submitting for each sample. Sequencing samples with template and primer must be in deionized sterile water or completely dried down. Upon arrival at NGC, all samples moved to a sequencing processing plate, dried down, and resuspended in reaction master mix. The minimum volume for samples is 5uL and the maximum is 25uL. If you sample is less than 5uL please add deionized water to bring it up to 5uL. If the volume required is more than 25uL please concentrate the sample. If the sample has been dried down, enter "dry". Primer Each sample is required to have a single primer. A system primer or a custom primer can be selected. If Custom is selected, a dialog box will appear for you to enter the primer name. If the dialog box does not appear, check to make sure you do not have pop-ups blocked while working in dnatools. If a system primer is selected the primer name will be automatically entered into the Primer Name text box. Do not manually edit the name in the Primer Name text box. Legal characters are: a-z, A-Z, 0-9, and - Illegal characters are any other characters. Do not use underscore, period, or any quoting characters. There are numerous convenient tools to assist you with filling in the primer names. Click the the options are described. and all Finally, you need to fill-in your primer concentration. If you are unsure as to what primer concentration is appropriate for your template, please NGC webpage for guidelines. Once you have filled in the boxes, click the submit key in the middle of the page. After you click submit and the request is accepted, a summary table is displayed. The first field of the table is the Order Number. 5

6 Order Number This is the number you will put on the sample tube: Please print out 2 copies of this summary page. Send one with your samples and keep the other copy for your records. Note, you need only label your tubes with the Order # and sample number. It is vital that the number on the sample tube match the number on job request in order for the proper sample name to be assigned to the data. Please write the Order Number on each strip tube or plate. Please write the sample number on each tube in the strip. 6

7 Uploading a Sequencing Request Your second option for entering a request is to Upload and Import Files. The blue Upload and Import File enables you to upload sample names from an excel file. You can use the Upload and Import File option for anywhere from 1 to 95 sequencing samples. Clicking on the Upload and Import File link will take you to the following page: 7

8 The first step is to save the Sequencing Template file to your hard drive. You need only do this once and then each time you are submitting samples you can fill-out the template, save that file with a specific file name or date, and then upload the completed file. Thus, once the template is saved to your hard drive you have it ready each time it is needed. The sample and primer naming has the same legal character restrictions as when you enter a sequencing request (a-z, A-Z, 0-9, and -). As when you enter a sequencing request, you need to list the amount, in nanograms, of template DNA, the liquid volume of the sample, the type of template (plasmid, PCR, BAC ), and the size of the template DNA in base pairs. When you have completed filling out the template form save it to your hard drive as a tab-delimited file. Open dnatools and go to the Upload and Import link. You will begin by selecting the Payment Type. The payment options are Purchase Order, Account Name, or credit card. When you select your payment option a dialog box will come up so that you can enter the PO, Acct Name, or credit card. If the dialog box does not appear, check to make sure you do not have pop-ups blocked while working in dnatools. If entering credit card information note that must be hand written on the printed form never type that information into the server. UNR campus users should choose Acct Name and enter the PI s last name. If your UNR lab uses more than one PCard then please also add either the last 4 digits of the PCard number or enter the name of the account that you have established with us in the comments field. Select the service type you wish from the drop-down list. Select whether you have already added the primer or not, and if there is primer in some, but not others you should make a note of that in the comments box. Analysis Type refers to Fragment Analysis requests and can be ignored for entering sequencing requests. Now, use the browse function at the bottom of the page to find the tab-delimited Excel file you created and open that file so that it is listed in the Upload File box. Finally, submit the request. After you click submit and the request is accepted, a summary table is displayed. The first field of the table is the Order Number. Please print out 2 copies of this summary page. Send one with your samples and keep the other copy for your records. Note, you need only label your tubes with the Order # and sample number. It is vital that the number on the sample tube match the number on job request in order for the proper sample name to be assigned to the data. Please write the Order Number on each strip tube or plate. Please write the sample number on each tube in the strip. 8

9 Viewing your Orders The User Queue Viewer allows you to view both the unprocessed and processed jobs. If you have a job in the Unprocessed Orders queue you can click on the order number and either view or delete the order. Once your samples have been added to an electronic run plate the order will appear in the Your Orders in Plates table and they cannot be deleted at this point. Please note that your samples may be in processing prior to appearing on an electronic run plate. Finally, once the order has been fully processed and the data is up the server the order will appear in the Your Processed Orders table and you can click on the order number to view the results table. Alternatively, you may select the View Your Requests, in the upper left-hand Sequencing box, to display a summary table of all sequencing requests. The sequencing request queue can be searched by Request or Order Number. This form also allows you to delete requests and orders. 9

10 Deleting an Order or Request If you have a job in the Unprocessed Orders queue you can click on the job number in the User Queue Viewer and then click the big red Delete Job button to delete the job. Alternatively, to delete an order or only a few samples within an order, begin on the menu page and select View Your Requests. This will take to you a link which contains your sequencing orders and requests. Note: you can only delete samples or orders which are pending processing. Once processing begins, you cannot delete the order or request. If you wish to delete an order, the first thing to do is select that order from the list of your orders found in the box. Then select the Delete Order button at the bottom of the box. A pop-up box will appear telling you that you are about to delete an order. Select OK if you do wish to delete the order. If the pop-up box does not appear, check to make sure you do not have pop-ups blocked while working in dnatools. Finally, you click submit and the order will be deleted. To delete a sample from an order, highlight the order and then select the View Order option. This will take you to a screen which lists each sample in the order and shows you the requisition number for each sample. Click on the requisition number of the sample you wish to delete and that action will take you back to the View Sequencing Requests page with that sample s requisition number now appearing in the top box. Select the Delete Request option button. Click submit and the sample will be deleted from the order. 10

11 Display Order Summary This link will allow you to view and print any of your orders. You may search for an order by: 1.) Current Orders in the Queue: This is a list of the orders that are pending processing. 2.) This Month: This will display a list of all the orders you placed in the current month. 3.) Select Date: This allows you to select the month and year and see a list of the orders that were placed in that month. Select how you wish to view the list of orders and then submit. A Summary Table will be display your orders. You may select the View button for any order and the summary page of that order will appear. 11

12 Viewing and Downloading Your Sequencing Results To view or download your DNA Sequencing Results select the Download DNA Results link. Two tables are displayed, so you can choose the data you want to view either by Order Number or by the Run Plate Number. After you make your selection click submit. The next page allows you to either download or view you sequencing results. 12

13 Viewing the Chromat To view chromats you must first download the.ab1 files and then open the.ab1 file(s) using software that will allow viewing of.ab1 files. If you have FinchTV, that will allow you to open and view the files; however, Finch TV is no longer supported or available to download. Current freeware options include: 1.) Technelysium s Chromas is a free, simple, easy-to-use viewer for Sanger sequencing chromatograms. Free download: 2.) SnapGene offers freeware for Windows, macos, Ubuntu Linux, or Fedora Linux devices to view Sanger sequencing chromatograms. Free download: You have two options for downloading the.ab1 files either all the files within a single job request or individual files. If you want to download all of the.ab1 files for a job, click the Chromat button in the Sequencer Output box and this will download all of the.ab1 in a single zipped file. That zipped file can then be unzipped and the.ab1 files viewed. Alternatively, to view an individual chromat, click the Chromat link to the left of the View Text box. This will download the.ab1 file and that in turn may be opened using your choice of viewing software. Once you have downloaded the.ab1 file and opened it in your choice of freeware, view the chromatogram, but also be sure to view the quality values. The quality values, or Q20 values, are displayed in bar graphs above or below each base call. A shorter bar indicates a lower, poorer Q20 value, while a taller bar indicates a higher, better value. Using the SnapGene software, when holding the cursor over a base a small pop-up box will appear that lists the base call, base number, and quality (example shown below). The quality call is the Q20 value for that particular base. The term Q20 value came about because a quality call of 20 indicates an accuracy of 99% for the base called. The base-calling program used in conjunction with the ABI Prism 3730 DNA analyzer is Phred, a program developed by Dr. Phil Green and Dr. Brent Ewing. Phred reads DNA sequencer trace data and calls bases. It also assigns quality values to the bases, and writes the base calls and quality values to output files. After calling bases, the program evaluates the trace surrounding each called base using four quality value parameters to quantify the trace quality and these quality scores range from 4 to about 60, with higher values corresponding to higher quality. 13

14 G44 Quality: 52 To View the Text Calls Under the column View if you select the View Text link it will take you to a new page. Above the text box there are four buttons relating to the text string of sequence. The important button here is the Trimmed Sequence button. This displays the sequence that has good Q20 values and is thus good sequence. The trimming program trims off at points where the Q20 drops below 20 for 8 or more bases. Occasionally, when the signal intensity is low, the unincorporated dye coming through will cause a drop in Q20 for more than 8 bases and thus the text is trimmed at that point (around 60bp), while there is still good sequence following the dye front. In this case, you could use the Phred Sequence button that will show you the raw, un-trimmed data. Trimmed Sequence 14

15 Downloading Your Sequencing Results Download ALL files Download a single file The top table provides various options for downloading the data for all the samples in the order. You have the option to download text files, the chromatograms, the quality data or the trimmed sequences and the options and the files that will download for each option are listed in the table below. When you select the desired button, a pop-up window will open. Select Save and save the file to your hard drive. The files will be compressed files and will need to be unzip before you can open the files in Word, Excel, or a chromat viewer. With the buttons at the top of the page you may download ALL of the samples for that job order in a single folder. If you wish to download the data for an individual sample, use the Text or Chromat link under the Download column. Heading Button / Option File Opens in Trimmed/ Untrimmed Files for each sample What is in the file Sequencer Output Text.seq Word U all separate only Untrimmed seq. Chromat.ab1 various U all separate chromat with everything Phred Output fasta.seq Word U all separate name & Untrim seq. qual.qual Excel all separate name and Q20 phred.phd1 Excel all separate name, base, Q20, and.. Trim Output Trimmed.seq Word T all separate name & Trimmed seq. Submit.seq Word T all separate name & Trimmed seq. Download Text.seq Word U single only Untrimmed seq. View.ab1 various U single chromat with everything Create Fasta Library Phred/Fasta.txt Excel U all in 1 file name & Untrim seq. Trimmed.txt Excel T all in 1 file name & Trimmed seq. If you wish to have a single file containing all the samples for the job order, then you need to return to the Menu page and use the Create Fasta Library. 15

16 Creating a Fasta Library If you want to download all the samples within a job request as a single file then you select the Create User Nucleotide Fasta Library link from the menu page. This will take to the Create User Fasta Library page. Select the plate that you wish to put into the library, name the library or use and existing name, select the source, append the sequences into the library, and submit. 16

17 Search Sequencing Results If you wish to search for some sequencing data you would use the Search Sequencing Results link on the menu page. A page will open which will allow you to search by sample names and/or primer name: Select the year and then enter a sample name in the Sample name text box. Select the type of character matching to use: IS LIKE or IS. For example, if you select IS LIKE and enter s for the sample name and it will display any sample name beginning with s. Or, select IS and enter s1 and it would display sample names that are exactly s1. This method may be used in conjunction with primer names. The search tool allows you to specify either AND or OR based matches. OR will return matches if either the sample OR primer name match the search criteria. If the AND radio button is selected, both search criteria are required to match. 17

18 PI Group Results View You may view the data that other researchers in your lab have produced by selecting PI Group Results View. Step #1 Step #2 Step #3 You need to select whether you wish to view DNA Sequencing Data or Fragment Analysis Data, and then select the name of the individual who submitted the job. This will take you to the Download Results page and you may continue as described above. 18

19 Job Quality Report The Job Quality Report link will take you to a page where you will see all of your processed jobs. 19

20 Select the job and then submit and this will take you to a table listing the quality calls for that job. This table lists the sample name, primer, trimmed read length, average Q20, and average signal strength which are quality calls you can use in troubleshooting your sequencing reactions. Please visit our website (genomics@cabnr.unr.edu ) Sanger Sequencing page for discussion on how to use these quality calls in troubleshooting as well as tips on how to improve your sequencing reactions. 20