MASSIVELY PARALLEL SEQUENCING SERVICES

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1 OCTOBER 18, 2017 MASSIVELY PARALLEL SEQUENCING SERVICES McGill University and Génome Québec Innovation Centre User Guide: Illumina sequencing technologies Version 5.9 Copyright 2015 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 DNA SAMPLE SPECIFICATIONS... 4 ACCEPTED FORMATS FOR DNA SAMPLES/LIBRARIES... 4 SEQUENCING-READY LIBRARIES/AMPLICONS AND THEIR COMPLEXITY... 5 GUIDELINES FOR RNA SAMPLES... 6 IMPORTANT GENERAL CONSIDERATIONS... 6 RNA SAMPLE SPECIFICATIONS... 7 SAMPLE SUBMISSION... 8 PREPARING SAMPLES FOR SHIPMENT... 9 SHIPPING ADDRESSES FOR SAMPLES

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: o has an OD 260/280 ratio of 1.8 to 2.0 o has an OD 260/230 ratio of at least 2.0 o does not contain insoluble materials, RNA, chelating agents, divalent metal cations, denaturants, detergents or carry over contamination from the starting organism/tissue perform DNA extractions with commercial kits rather than homemade solutions, perform a cleanup step prior to submitting samples, resuspend DNA samples in 10 mm Tris-HCl ph 8.0 with 0.1 mm of EDTA (higher concentrations of EDTA will inhibit some enzymatic reactions), quantify samples using fluorometric-based methods rather than purely spectrophotometric-based methods (e.g. Nanodrop) which tend to overestimate sample concentrations, resulting in an inadequate amount of starting material, assess integrity on agarose gels, ensure that the amount and concentration of each sample be within the range specified in the DNA Sample specifications section, measure precisely and report the volume of each sample contained in each well using the accompanying Service Request Form. 3

4 DNA Sample specifications Should the sample volume be inferior to the minimum volume specified in the table below, it will either be diluted to an appropriate volume without prior client consent or will be outright refused. Maximal DNA concentration should not exceed 150 ng/l. A volume of 3 µl is used for DNA Quality Control. Table 1. Summary of DNA sample requirements Source of nucleic acid Library Type Required Quantity (ng) Required Volume (µl) Shotgun (short inserts) to 50 Shotgun with gel extraction (short inserts) to 50 PCRfree (TruSeq) to 50 3, 5 or 10 kb Mate Pair to 50 WGBS to 50 Target Capture (SureSelect) to 50 Target Capture (Roche Nimblegen) Methyl Capture (Roche Nimblegen) to to 50 ChIP DNA ChIP-Seq to 30 Sequencing-ready Library to 75 Fragment size range of ChIP DNA or amplicons: bp. Sequencing results are provided on an as-is basis. Library size should take into consideration: 1) Adaptors add ~120 bp to fragment lengths; 2) the number of sequencing cycles If replacement samples are needed, please send the full amount required, not top ups. Additional sample QC fee will be applied for each replacement sample. 4

5 Accepted formats for DNA samples/libraries Samples/libraries must be submitted in 96-well plates regardless of the number of samples. There should be one sample per library type per well. 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# VWR Aluminum Foils for PCR and Cold Storage, Cat# Pipelines have been optimized for high throughput processing of samples. Introducing plates other than the two models specified above may lead to sample loss and damage to the robotic liquid handlers. Therefore, samples submitted in non-conforming plasticware will be re-plated in the proper plates. There will be additional fees for re-plating them. If samples from multiple projects are submitted at once, they have to be on different plates. The requirements mentioned above also apply for QC only projects. Sequencing-ready Libraries/amplicons and their Complexity The Illumina software that delineates the cluster boundaries on the flow cell and carries out the base calling is dependent upon sequence complexity at the ends, particularly the first dozen or so base pairs, at either end of the inserts being sequenced. For this reason, any type of library which does not exhibit normal or near-normal sequence complexity in these regions must be brought to our attention otherwise the sequencing data will be less than optimal. This includes but is not limited to amplicons, reduced genome representation methods such as Restriction site associated DNA (RAD) marker libraries, and libraries with reduced nucleotide complexities such as bisulfate-converted libraries. To overcome this issue with low complexity libraries or libraries with low complexity at the ends of the inserts, the complexity can be indirectly increased by spiking in a complex library (such as the control phix174 library supplied by Illumina or one which will generate useful reads for the client s research) at 10-50% of the lane, depending on the complexity of the initial library. The same nucleotide complexity issue described above applies to the index sequence when multiplexing samples. It is not recommended to multiplex only two indexed libraries together as this does not generate enough nucleotide diversity in the index read. For best results, a minimum of 3 indexes should be used per lane when multiplexing samples. Please note that Rapid Mode sequencing is done by default using the dual flow cell option. This means that all the libraries to be sequenced on the run must have different barcodes. If the barcodes of the libraries to be submitted are not unique, the single flow cell option can also be done. However, this requires that an additional cost for a Offboard flowcell prep addon item be added to the quote/order. 5

6 Guidelines for RNA Samples Important General Considerations When working with biological samples that are destined to be submitted for RNA profiling (e.g. RNA- Seq, small RNA-Seq), it is recommended to: wear new gloves and keep samples on ice. resuspend samples in commercial RNase-free water. avoid DEPC-treated water. avoid resuspending samples in buffers containing detergents (e.g. SDS) as these will inhibit the enzyme reaction used to synthesize cdna. If Trizol is used to extract RNA samples, it is recommended to perform a final clean-up (for example, with Qiagen mrna clean-up kit) prior to submission. Sample identification of the tubes must correspond exactly to what is specified in the Request Form. Aliquots must be sent, not stock samples. All RNA samples must be submitted in 1.5 ml Eppendorf tubes. Any amount of remaining sample material will be retained for a short period of time after the requested service has been completed and will then be discarded. 6

7 RNA Sample Specifications Total RNA is used as starting materials for all the protocols. A volume of 2-4 µl is used to first assess RNA Quality Control. Quality Control is performed on total RNA, even for samples for which small RNA profiling will be performed. This means that the Platform cannot account for the presence/absence of small transcripts in the sample. It is therefore not guaranteed that a library will be generated from a sample which has successfully passed the Qc. It is better to dilute concentrated samples (e.g., 1 µg/µl down to 100 ng/µl) and provide the minimal volume required rather than submitting samples with higher concentration but smaller volumes. Table 2. Summary of sample requirements for the construction of libraries from RNA Application Amount (ng) Minimal concentration (ng/µl) Volume (µl) RIN* Eukaryotic gene expression (protein coding transcripts) ~ >6.5 Prokaryotic gene expression ~ >6.5 Meta-transcriptomics species) (multiple ~ >6.5 Coding and long non-coding transcripts profiling ~ >6.5 Small RNA profiling** (<200 nt) ~ >6.5 *RIN = RNA Integrity Number; assessed on a Bioanalyzer (Agilent) ** The protocol takes advantage of the 5 -phosphate and 3 -hydroxyl group of mature mirna. The presence/absence of other small transcripts cannot be guaranteed. 7

8 Service Request and Sample Submission Service Request Form and Sample Submission (Creating a New Request) The new Request Form functionalities are used for new projects only. Login to your Nanuq account here. 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 and Sample Submission (Existing Request) Login to your Nanuq account here. 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, 2) in house reception of samples previously submitted for Qc only or 3) 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. Please note that since samples will be entered and processed in the same order as in the Sample Submission Sheet, it is strongly recommended to that, for RNA-Seq, Methyl-Seq and ChiP-Seq projects, to randomize samples according to their experimental condition to minimize the technical variability of the sequencing. Due to the large numbers of samples that are processed at the Centre, we cannot guarantee that specific loading schemes can be honored; we reserve the right to sequence lanes over multiple runs as deemed appropriate and without prior notice. Specific schemes have to be entered in the Comments column of the Sample Submission Sheet. 8

9 Preparing Samples for Shipment The shipment must include a printed and signed copy of the complete Service Request Form. DNA and RNA samples and sequencing-ready libraries should be sent on dry ice pellets (not large blocks), unless otherwise stated. The tubes (RNA) and/or 96-well plates (DNA and sequencing-ready libraries) should be sealed in a Ziploc-type plastic bag. If submitting many tubes (RNA), they should be neatly organized in a rack or cardboard box. It is recommended to authorized personnel prior to bringing samples at the Innovation Centre. Samples crossing the Canadian border should be shipped early in the week. 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. 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 thrown out (libraries will be kept for at least 1 year). Shipping Addresses for Samples Complete shipping addresses are listed below by nucleic acid type: DNA / RNA samples: Attn: MPS Services c/o Sylvie LaBoissière McGill University and Génome Québec Innovation Centre, Suite , Dr.-Penfield Avenue Montréal (Québec) Canada H3A 0G1 Ready-to-sequence libraries: Attn: MPS Services c/o Alexandre Montpetit McGill University and Génome Québec Innovation Centre, Suite , Dr.-Penfield Avenue Montréal (Québec) Canada H3A 0G1 9