How to submit samples

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How to submit samples This document is a step by step guide explaining how to submit samples. It includes the following topics: 1. How to access the Sample Submission Form from Nanuq 2.

1 How to submit samples This document is a step by step guide explaining how to submit samples. It includes the following topics: 1. How to access the Sample Submission Form from Nanuq 2. How to fill in the Sample Submission Form 3. How to submit the Sample Submission Form in Nanuq 4. How to track a submission in Nanuq 5. What tube and plate types are acceptable 6. Sample preparation specifications 7. Sample and primer organization 8. How to send samples to the Sequencing Service It is important to follow the instructions described in this document in order to avoid processing delays. Step 1 - Accessing the Sample Submission Form from Nanuq 1. Enter the following address: 2. Enter user name and password which was issued upon account opening.

2 3. Click on Sequencing to access the Sequencing Platform Menu. 4. Select Submit Samples for sequencing within 2 to 4 working days or Submit Samples in BaseXpress mode for sequencing within 24 hours.

Indicate whether or not the SNP (Single Nucleotide Polymorphism) discovery service is required.

3 5. Select the type of service required; sequencing or DNA preparation for the extraction and sequencing of plasmid DNA from bacterial colonies. 6. Indicate whether or not the SNP (Single Nucleotide Polymorphism) discovery service is required. This service includes sequencing, data analysis and the production of a report. Additional information about this service is provided on the website.

4 7. Indicate the type of container that the submitted samples will be in. A 96-well plate is required for all submissions totalling 48 samples or more. 8. Click on Download Excel Template.

5 9. Open the Sample Submission Form in Microsoft Excel or OpenOffice. Step 2 - Completing the Sample Submission Form The information provided in the Sample Submission Form is necessary for the proper and efficient sequencing of samples. It is important to read the header in the Sample Submission Form in order to: verify that the correct form is being used, access instructions to complete the form, be aware of the required sample concentrations and volumes, access the list of primers provided by the Sequencing Service, be aware of the permissible tube and plate models.

Plate or Tube Set Name field A name must be given to each plate or tube set. This name must be unique and cannot exceed 50 characters.

6 1. Purchase Order Number field A purchase order number obtained from your institution s accounting department must be entered if the payment option chosen was purchase order number. Note that this option was chosen when the account was opened. 2. Plate or Tube Set Name field A name must be given to each plate or tube set. This name must be unique and cannot exceed 50 characters. In addition to alphanumeric characters, the hyphen ( - ), underscore ( _ ) and period (. ) are permissible however accents and blank spaces will not be accepted.

7 3. Sample name and Primer field Names cannot exceed 50 characters. In addition to alphanumeric characters, the hyphen ( - ), underscore ( _ ) and period (. ) are permissible however accents and blank spaces will not be accepted. Sample Names Sample names must be entered in the column entitled Sample name in the following order: A01 to A12, then B01 to B12, etc. and not A01, B01, C01, etc with empty rows between the sample names. The names must be unique. If you would like the primer name to appear in the chromatogram file name (i.e., the file ending with the extension ab1) then it must be included in the sample name. Primer Primer names must be entered in two places Primer names must be entered in the Primer column. Only one primer is permitted per cell. If a sample is to be sequenced with more than one primer then that sample name must be entered in the Sample name column as many times as it needs to be sequenced. Then all associated primers must be listed in the Primer column. The purpose of this is to indicate which primer must be used to sequence the sample listed in the Sample name column. See example in the screen shot below. Primer names must also be entered in the Sample name column. For this primer name list ONLY the project name and Type of DNA columns must be filled-in. The purpose of this is to provide the technicians with the physical location of the primers. Primer : Enter only one primer per cell. Sample name : Enter a unique name for each sample. Enter the required primer name for this sample in the column entitled Primer.

8 4. Chose the appropriate DNA type from the drop-down list in the Type of DNA column. Type of DNA : If the type of DNA is primer, only the Sample Name and Project Name columns are required.

9 5. Enter the PCR product or plasmid size in the Size of template (base pairs) column. 6. The project name is case sensitive therefore it must be entered exactly as it appears on the Homepage.

10 7. Additional information pertinent to sequencing may be entered in the Information on DNA column. Download sample files [Excel] Sample Submission Form sample and primer organization in a 96-well plate [Excel] Sample Submission Form - sample and primer organization in PCR strip tubes Complete and save the file in an easily accessible folder. Step 3 Submitting the Sample Submission Form in Nanuq All samples to be sequenced must be submitted electronically through Nanuq. Return to the page entitled Sequencing Samples Submission and follow the instructions described in Step 3- Upload Excel File on Nanuq s webpage.

12 Check mark the box BaseXpress for all samples to be sequenced within 24 hours. For more information about the BaseXpress service, most notably the latest acceptable drop-off time and price, it is important to read the document entitled What is the BaseXpress service?.

Nanuq will then validate the submission as described below in step 4. 1. Sample validation FAILED! Nanuq will list all the errors found.

13 Nanuq will then validate the submission as described below in step Sample validation FAILED! Nanuq will list all the errors found. Once the errors have been corrected, save the file and click on Upload the same file again. Errors listed as 1 and 2: These errors were generated because the Type of DNA chosen in row 23 was primer. Nanuq understands this to mean that the entry in sample name (i.e., PCR2_P1) is the primer name and does not expect to find an entry in Primer column nor Size of template (base pairs) column. To correct this error change the entry in Type of DNA from primer to either Not purified PCR or Purified PCR, whichever is appropriate.

If the project name originally included capital letters they must appear in this entry.

14 Error listed as 3: This error was generated because the project name in row 33 does not appear exactly as it was written on the Sequencing Services Request Form. If the project name originally included capital letters they must appear in this entry. To verify the project name return to your Homepage on Nanuq. To correct this error change the entry in Project name from endymion to Endymion.

Nanuq expects to find a primer name listed in the Sample name column.

15 Error listed as 4: This error was generated because there is an entry in Project name and Type of DNA columns however there is no entry in Sample name of row 37. Nanuq expects to find a primer name listed in the Sample name column. To correct this error list the name of the primer in the Sample name column. Do not hesitate to contact us if any problems should arise.

2. Validation successfully completed! Verify that the summary information is accurate, specifically the total number of samples and information entered into the Excel sheet.

16 2. Validation successfully completed! Verify that the summary information is accurate, specifically the total number of samples and information entered into the Excel sheet. To complete the submission click on Confirm Sample Submission. If this is not done the submission is not logged in Nanuq. In order to make a correction, return to the Sequencing Samples Submission page by clicking on Cancel. The sample submission confirmation page, which follows, includes the submission number generated by Nanuq. This submission number is very important as it may be used to follow the progress of your samples and will be needed as a reference for all submission inquires.

17 All questions with respect to a submission are directed to the contact person listed on this page. It is therefore important to confirm the accuracy of this information. From this page it is possible to : Print the waybill. It is mandatory to include this waybill with the samples. Submit new samples View the submission lot

18 The bar code is scanned once the samples have arrived to the sequencing lab.

19 BaseXpress must appear on the waybill in order for the samples to be processed within 24 hours.

This page lists all submissions and associated information, namely, number of samples, their

20 Step 4 Tracking a submission in Nanuq The Sample Submission Search page is available from the sequencing platform menu. This page lists all submissions and associated information, namely, number of samples, their status, plate name, etc.) Additional submissions may be made from this page. To view submission details on the Samples Submission Lot page click on the sample submission lot number.

21 From this page it is possible to : Print the waybill. Remove the submission by clicking on Delete Lot. This function remains available until the sequencing group accepts the samples. View the submission status by clicking on the view samples icon. o Samples will remain logged as waiting for approval till they get to the sequencing laboratory. o Samples are logged as accepted the day of their reception. o Samples will be logged as rejected if the submission requirements are not followed or if they arrive in poor condition o Samples will be ignored upon your request. o Samples results are available on Nanuq for those logged as sequenced. Step 5 - Acceptable tube and plate formats Only certain plate and tube models are compatible with the Sequencing Service procedures. These accepted plate and tube models are: PCR strip tubes PCR 96-well plates PCR 384-well plates Suggested products : PCR strip" tubes: UltiDent Scientific, 0,2 ml thin-walled 12 tube and domed cap strips, catalogue number: AB-1113 PCR 96-well plates: Axygen Scientific, 96-well half-skirt PCR microplate clear, catalogue number: CA PCR 384-well plates: Corning, Thermowell Gold PCR Plates, catalogue number: 3757 Plate sealers: Applied Biosystems, MicroAmp Clear Adhesive Films, catalogue number: A 96-well plate must be used for all submissions of 48 samples or more.

Step 6 Preparing Samples Volume and concentration required per sequencing sample: Volume Concentration Unpurified PCR 20 μl minimum PCR amplifications must be verified on an agarose gel and Purified

22 Step 6 Preparing Samples Volume and concentration required per sequencing sample: Volume Concentration Unpurified PCR 20 μl minimum PCR amplifications must be verified on an agarose gel and Purified PCR 7.5 μl minimum a copy of the gel picture must be sent to the sequencing service with the samples. PCR products of less than 250 bp result in lower quality sequences due to an oversaturation phenomenon whereby they appear much more intense than usual and to the compression of bases at the beginning of the sequencing read, which is an inherent limitation of the 3730xl technology. PCR products greater than 2000 bp can be difficult to sequence because the concentration of DNA submitted is generally too low. The longer the PCR products the higher DNA concentrations are required to achieve good quality sequencing. Plasmid DNA or phage BAC/PAC end sequencing Volume 7.5 μl minimum ng/μl 20 μl minimum 500 ng/μl minimum Concentration

23 Step 7 Organizing Samples Samples must be organized in the plate/tubes in precisely the same way as they were in the Sample Submission Form. The tubes must be identified using their well position from the Sample Submission Form: A01, A02, A03 and not their sample names nor primer names. Samples and primers must be aliquoted in separate wells as many times as they will be used even if a sample or a primer is to be used multiple number of times. The primers must be aliquoted precisely in the same order that the samples, they are to be used with, are in. Step 8 Sending Samples The samples with their WayBill must be sent by mail or delivered in person between 9:00 AM and 5:00 PM, Monday to Friday. BaseXpress service samples must arrive to the Sequencing Service before 11:00 AM, Monday to Friday. Samples must be addressed to: Sequencing Service McGill University and Génome Québec Innovation Centre 740, Dr-Penfield Avenue, Room 7300 Montréal, QC, H3A 0G1 Tel: , ext Fax: Do not hesitate to contact us if any problems with the sample submission should arise: By sequencing.service@mail.mcgill.ca By telephone: ext

SEQUENCING SERVICES. McGill University and Génome Québec Innovation Centre FEBRUARY 28, Version 3.5

SEQUENCING SERVICES. McGill University and Génome Québec Innovation Centre FEBRUARY 28, Version 3.5 FEBRUARY 28, 2018 SEQUENCING SERVICES McGill University and Génome Québec Innovation Centre Sanger Sequencing User Guide Version 3.5 Copyright 2010 McGill University and Génome Québec Innovation Centre