DECEMBER 17, 2018 MASSIVELY PARALLEL SEQUENCING SERVICES McGill University and Génome Québec Innovation Centre User Guide: Illumina sequencing technologies Sequence-ready libraries Version 6.0 Copyright 2018 McGill University and Génome Québec Innovation Centre All rights reserved.
Table of contents TABLE OF CONTENTS... 2 GUIDELINES FOR DNA SAMPLES... 3 IMPORTANT GENERAL CONSIDERATIONS... 3 SEQUENCE-READY SAMPLE SPECIFICATIONS... 3 ACCEPTED FORMATS FOR DNA SAMPLES/LIBRARIES... 4 SEQUENCING-READY LIBRARIES/AMPLICONS AND THEIR COMPLEXITY... 5 SERVICE REQUEST AND SAMPLE SUBMISSION... 6 SERVICE REQUEST FORM... 6 SAMPLE SUBMISSION FORM... 6 HOW TO FILL THE SAMPLE SUBMISSION SHEET... 7 PREPARING SAMPLES FOR SHIPMENT... 8 ANNEX 1: ADDITIONAL INFORMATION AND DEFINITIONS... 10 2
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 libraries for sequencing with the Illumina technology, it is recommended to: quantify library concentration using a qpcr -based method rather than purely spectrophotometric- (e.g. Nanodrop) or fluorometric-based (e.g. picogreen) methods to ensure the presence of sequences required for sequencing, ensure that the amount is within the range specified in the Sequence-ready sample specifications section, measure precisely and report the volume of each sample contained in each well using the accompanying Service Request Form. Ensure that plate identification corresponds exactly to what is indicated on the Request Form. Sequence-ready 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. A volume of 5.5 µl is used for Library Quality Control. Volume exceeding 110 µl will be discarded. Library concentration should not exceed 70 ng/µl. Libraries that are too concentrated will be diluted and Qc redone. If replacement samples are needed, please send the full amount required, not top ups. Additional fees will be applied for the QC of each replacement sample. 3
Table 1. Summary of library sample requirements Sequencing Type Required concentration (ng) Required Volume (µl) HiSeq X / HiSeq 4000 1 ng/µl 25µL MiSeq 0,5 ng/µl 25µL iseq 100 0.1 ng/µl 25µL NovaSeq 6000 (1 individual library ou pool / sequencing lane) NovaSeq 6000 (2-6 individual libraries or pools / sequencing lane NovaSeq 6000 (7 or > individual libraries or pools / sequencing lane) 5 ng/µl 50µL 2 ng/µl 30µL 1 ng/µl 25µL 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# 951020401 PREFERRED Corning Thermowell GOLD, Full Skirt, Cat# 3752 SECOND CHOICE We recommend the following sealing films: Life Technologies MicroAmp Clear Adhesive Film, Cat# 4306311 VWR Aluminum Foils for PCR and Cold Storage, Cat# 60941-074 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. 4
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 add-on item be added to the quote/order. 5
Service Request and Sample Submission Note that the work in the laboratory will only start when all required documentations is provided. Service Request Form 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. Incomplete Request Form are accessible in future sessions using the Request List option. The Request Form will remain on a Draft status until the Submit button is used. Draft Requests cannot not be processed and delays should be expected. The Client Management Office can only approve submitted requests. Sample Submission Form Login to your Nanuq account. In the Request Section, click Request List and choose the right one. To submit new samples, go to the Sample Submission Section and download the template file for submitting new samples. To review a submitted file, click on the file name already submitted. To add comments on samples, click on the file name already submitted, then on the Comments button. 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. 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. 6
How to fill the Sample Submission Sheet From the Request Form in Nanuq, click on add new samples to the request. Click No to the question Does your samples need extraction? Choose sequence-ready libraries from the Sample Category dropdown menu. Choose the proper container (columns or rows) depending on how your libraries have been/will be plated on the 96-well plate which will be submitted to the Platform. Note that this choice cannot be modified afterwards. Fill the template using the following instructions/information: Plate Type+: choose the proper layout (column or row) Plate Name+: write the name; avoid special characters. Adapter/Index type+: select the sequencing adaptor used during library preparation from a dropdown menu. Adapter names are from commercial kits. See Annex I for details on adaptors and indices. Do not hesitate to contact your Client Management Office representative for help if you believe that your adapters might be custom. Sample Type: individual library or pool. For the latter option, note that the first line contains information on the pool such as volume and concentration whereas subsequent lines define each library that is part of the pool. New samples or pools can be added one after the other in the template. Client Pool Name: name of the pool in Nanuq. Nb libraries / amplicons in pool+: number of libraries present in the pool. Client Library Name: name of each library in the pool. Seq Technology Library Type +: select from the dropdown menu Species: species used to construct the library Cohort: optional; 7
Fragment / Library Size (bp); size of the pool or individual library (insert + adaptor) Concentration and Volume: values must fit with requirements mentioned previously Type of Sequencing: select from the dropdown; refer to the quote from the Client Management Office. Number / Fraction of Sequencing Units+: fraction of the lane which will be attributed to the pool or library. For instance, 4 libraries sequenced on a lane will have 0.25 as fraction of lane. Index 1+: i7 index added to DNA fragment of interest during library preparation (see Annex I). Dropdown options are based on the choice of Adapter (line 22). If indices are absent from the dropdown menu, chances are that a wrong selection has been made when selecting the Adapter. Select another Adaptor and see if indices used appear in the menu. Index 2+: i5 index added to DNA fragment of interest during library preparation. See above for explanations. Contact the Client Management Office for questions on how to fill this Form. Once the file has been properly filled up, save and upload in Nanuq to submit it. Preparing Samples for Shipment The shipment must include a printed and signed copy of the complete Service Request Form and a copy of the Sample Submission Form. Samples plates must be sent on dry ice pellets. If the package contains heavy objects which could damage plates during transportation (e.g. ice dry blocks, ice packs), it is strongly recommended to protect plates from impacts. Custom sequencing primers (if required) should be sent in 1.5 ml Eppendorf tubes. Tubes must be properly identified. Primer concentration should be at 100 µm. The package must contain enough dry ice to ensure that samples remain frozen up to destination. Thawing during transportation could result in a loss of adhesion of the seal. This could lead to sample loss or cross contamination between samples. Samples crossing the Canadian border should be shipped at the beginning of the week to minimize the risks of having samples at the carrier s storehouse over the weekend in case of delays. 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. Samples can directly be brought to the laboratory. However, visits must be coordinated with the platform personnel beforehand. Opening hours are between 7H to 12H and 13H to 16H from Monday to Thursday and 13H to 16H on Friday. Only send aliquots from your samples. These will be kept 6 month after the completion of the service. They will be discarded unless the answer Yes has been selected for the question Would you like to have your original samples/primers returned to you? in the field Original Sample Disposal of the Request Form. 8
The shipping address is: Sequence-Ready Libraries MPS Services c/o Sylvie LaBoissière McGill University and Génome Québec Innovation Centre, Suite 5400 740, Dr.-Penfield Avenue Montréal (Québec) Canada, H3A 0G1 9
Annex 1: Additional information and definitions Library adapters are 58-64 nucleotides located at the extremities of the insert (black line). They contain: Sequences required for linking the library to the flow cell (P5 forward in red; P7 reverse in green) Sequencing primers (brown and gray) Index i5 (orange) and i7 (yellow) sequences Flow cell adapters: 25-30 nucleotides bound to the flow cell. Their sequence is complementary to library adapters. Flow cell adaptors are required for the sequencing of Read1 and Read2. Sequencing primers: 30-35 nucleotides. Required for sequencing read1 and read2 as well as index 1 and 2. Sequencing primers can be standard (present in the Illumina sequencing reagents) or custom. Index: generally 6-10 nucleotide long. Indices are required for assigning reads to the right sample. Index 1 or i7 index (or reverse) located on adapter P7 is read first and is usually the only one used during single read sequencing. Index 2 or i5 (or forward), is located on adapter P5 is read during paired-end sequencing. Note that this is distinct from in-line barcode which is not located in the adapter but within read 1 or read 2 or from molecular barcodes. In-line barcodes cannot be used to distinguish reads from various libraries sequenced together on a lane. Molecular barcode (UMI): 8-10 bases sequence added to the DNA fragment during the ligation step of library preparation. UMI are required for distinguishing independent DNA fragments from PCR duplicates. UMI are generally located just after Index 1. 10