MASSIVELY PARALLEL SEQUENCING SERVICES

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1 MARCH 16, 2014 MASSIVELY PARALLEL SEQUENCING SERVICES McGill University and Génome Québec Innovation Centre User Guide: Roche 454 sequencing technologies Version 4.2 Copyright 2014 McGill University and Génome Québec Innovation Centre All rights reserved.

2 Table of contents TABLE OF CONTENTS... 2 GUIDELINES FOR DNA SAMPLES... 3 IMPORTANT GENERAL CONSIDERATIONS... 3 SAMPLE SPECIFICATIONS... 4 ACCEPTED FORMATS... 5 INFORMATION ON AMPLICON SEQUENCING... 5 INFORMATION ON CDNA SEQUENCING... 6 SERVICE REQUEST AND SAMPLE SUBMISSION... 7 SERVICE REQUEST FORM (NEW REQUEST)... 7 SERVICE REQUEST FORM (EXISTING ORDER)... 7 PREPARING SAMPLES FOR SHIPMENT... 8 SHIPPING ADDRESS

3 Guidelines for DNA Samples Important general considerations The guidelines contained herein are aimed at providing you the best possible sequencing data within the quickest possible turnaround time. Any and all samples that do not conform to the guidelines expressed herein may be refused without compensation. When submitting nucleic acids for sequencing using next generation sequencing technologies, it is recommended to: submit samples of the highest possible quality and purity. resuspend DNA samples in nuclease-free water or in 10 mm Tris-HCl ph (no EDTA should be added as its presence may inhibit some enzymatic reactions). avoid resuspending samples in buffers containing detergents (e.g. SDS) or other additives that may inhibit enzymatic reactions during the library preparation and/or sequencing reaction. quantify samples using the PicoGreen method. Nanodrop or other purely spectrophotometricbased methods tend to overestimate sample concentration, resulting in an inadequate amount of starting material. ensure that the volume of each sample provided should be within the Sample specifications section. precisely measure and report the volume of sample contained in each well using the accompanying Service Request Form. Sample identification of the container(s) carrying the sample(s) must correspond exactly to what is specified in the Request Form. All correspondence regarding completed or ongoing services must refer to the corresponding quotation number (beginning with SCI ), or to the corresponding Nanuq Project Name. 3

4 Sample specifications Concentration values specified below are consistent with volume and concentration limits based on the manufacturer s protocols. Samples with concentrations inferior to those indicated below must be concentrated by the client prior to shipment. The minimum acceptable volume per sample is 30 µl. Should the sample volume be inferior to 30 µl, it will either be diluted up to a volume of 30 µl without prior client consent or will be outright refused. Table 1. Summary of sample requirements Source of Nucleic acid Library Type Quantity (µg) Concentration (ng/µl) gdna Shotgun 1 50 gdna gdna 3 kb Paired End kb Paired End Amplicon PCR with "Fusion primers" 0.45 ~15 Sequencing-ready Library Preparation of long insert libraries requires highly concentrated and pure DNA. The presence of any particulate matter or a high viscosity of the sample will interfere with the initial shearing step. This almost always results in the complete loss of sample material. The client accepts that sequencing results are provided on an as-is basis. The client will be informed of samples that fail the QC and can submit replacement samples. If replacements are needed, please send the full amount required as per the requirements, not top ups. Additional sample QC fee will be applied for each replacement sample. The client may decide to proceed with sub-optimal amounts or quality of sample, but accepts all risks. 4

5 Accepted formats Samples and libraries for 454 sequencing may be submitted preferentially submitted in 96-well plates regardless of the number of samples. Only two types of 96-well plates are accepted: Eppendorf twin.tec, Full Skirt, Cat# PREFERRED Corning Thermowell GOLD, Full Skirt, Cat# 3752 SECOND CHOICE We recommend the following sealing films: Life Technologies MicroAmp Clear Adhesive Film, Cat# We can provide such plates and seals to local users upon request. Please inform the client management office of such request upon submission of documentation for the requested service. Samples from multiple projects must not be submitted in the same plate, rather, a separate plate should be used for each project. All plates must be clearly labeled with the quote number, name of PI and date shipped. Samples or libraries may be submitted in 1.5mL, 0.5 ml or strip tubes clearly labeled if submitting 8 samples or fewer. Information on amplicon sequencing The quality of amplicon sequencing data is heavily dependent on the quality, purity and base diversity of the PCR products. Amplicon libraries should be prepared with "Fusion primers" as described in Roche's Amplicon Fusion Primer Design Guidelines which can be provided to Clients upon request. PCR products should be free of: - Unextended primers as these may form primer dimers and/or hairpins which can be amplification-competent and will consequently be used as templates for sequencing - Non specific background amplification products (e.g., seen as smears or ghost bands on agarose gels) Clients must take appropriate measures to ensure that the amplicons comply with the above and should provide supporting documentation such as agarose gel pictures or bio-analyzer traces. For amplicon cleanup, we recommend Ampure XP (Beckman Coulter). A protocol can be provided upon request. Low amplicon sequence diversity can be problematic when using the FLX+ chemistry. Please contact us before designing amplicons for this technology. 5

6 Clients have the option of choosing to pool their amplicons themselves or having the Innovation Centre pool their amplicons for them on a fee-for-service basis. The Innovation Centre cannot be held responsible for unequal representation of amplicon reads within a pool. Even with perfect equimolar representation of each individual amplicon in the pool, it is still possible that other sample-specific factors may influence the actual number of reads obtained per sample. Roche offers two chemistries for amplicon sequencing; Lib-A and Lib-L. The Lib-A chemistry and corresponding Lib-A fusion primer sequences is reserved for bi-directional sequencing only. For unidirectional sequencing, Lib-L chemistry and corresponding Lib-L fusion primer sequences should be used. Information on cdna sequencing The Roche GS-FLX sequencing technology is notorious for having difficulties in sequencing homopolymer stretches whereby the length of homopolymer stretches may be over- or underestimated in any given read. Moreover, DNA templates containing homopolymers may adversely affect the sequencing of neighboring DNA templates on the sequencing matrix (pico-titer plate). For the above reasons, the Innovation Centre reserves the right to refuse sequencing cdna preparations employing oligo-dt priming for the synthesis of the first strand of cdna. The preferred method for the synthesis of the first strand of cdna method is by random priming. 6

7 Service Request and Sample Submission Service Request Form (new Request) The new Request Form functionalities are used for new projects only. Login to your Nanuq account In the Request Section, click Add new Request and follow the instructions. Do not use the Back button of your Browser to go back to previous pages. Use the menu on the lefthand side of the screen. The Request Form is completed when the Submit button is used, otherwise, its status will remain as Draft. Incomplete new Requests will become accessible in future sessions using Request List (see below). The work in the laboratory will only start when all required documentation is provided. Service Request Form (existing order) Login to your Nanuq account In the Request Section, click Request List. This is used when 1) new samples or replacement samples are submitted as part of an existing project or 2) to continue entering out new information in a Draft Request. To submit new samples, go to the Sample Submission Section. From there, you can: Download empty templates for submitting new samples. Review previous sample submissions. Add comments about your samples. Do not add new samples or replacement samples to an existing template file. Do not use the Back button of your Browser to go back to previous pages. Use the menu on the lefthand side of the screen. 7

8 Preparing samples for shipment The shipment must include a printed and signed copy of the complete Service Request Form. Samples should be sent on dry ice, unless otherwise stated. The tubes and/or 96-well plates should be sealed in a Ziploc-type plastic bag. If submitting many tubes, they must not be simply dumped randomly into a bag, but rather should be neatly organized in a rack or box. Samples may be dropped off in person, but drop offs must be coordinated with authorized personnel prior to bringing samples at the Innovation Centre. Samples crossing the Canadian border should be shipped early in the week as even expedited shipments may be held up at customs. All required documentation for customs must be duly included with the package. The use of clear phrases such as: Non-biohazardous biological sample, Purified DNA from [species], For research use only, and Of no commercial value will help expedite customs clearance, thereby avoiding unnecessary delay and risk of degradation. Take appropriate precautions to protect plastic ware containing samples from being crushed by shifting contents or external forces during shipment. Only send aliquots (in sufficient amounts), not stock samples. Any amount of remaining sample material will be retained for a period of approximately 1 month after the requested service has been completed and will then be destroyed and discarded (libraries will be kept for at least 1 year). Leftover samples can be returned to researchers upon special request. Such a request should be made to the client management office at the beginning of the project. All fees for returning the samples are the client s responsibility. 8

9 Shipping address The complete shipping address for shipping DNA destined for Roche sequencing is the following: Attn.: MPS Services c/o Pierre Lepage McGill University and Génome Québec Innovation Centre, Suite , Dr.-Penfield Avenue Montréal (Québec) Canada H3A 0G1 9

SEQUENCING SERVICES. McGill University and Génome Québec Innovation Centre MARCH 16, Version 4.0

SEQUENCING SERVICES. McGill University and Génome Québec Innovation Centre MARCH 16, Version 4.0 MARCH 16, 2014 SEQUENCING SERVICES McGill University and Génome Québec Innovation Centre SNP Validation and SNP discovery services Next-Generation Sequencing technology User Guide Version 4.0 Copyright