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MAY 1, 2015
MASSIVELY PARALLEL SEQUENCING SERVICES
McGill University and Génome Québec Innovation Centre
User Guide:
Illumina sequencing
technologies
Version 5.5
© Copyright 2015 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
DNA SAMPLE SPECIFICATIONS ................................................................................................... 4
ACCEPTED FORMATS FOR DNA SAMPLES/LIBRARIES........................................................................... 5
SEQUENCING-READY LIBRARIES/AMPLICONS AND THEIR COMPLEXITY ...................................................... 5
GUIDELINES FOR RNA SAMPLES ...................................................................................... 6
IMPORTANT GENERAL CONSIDERATIONS ........................................................................................ 6
RNA SAMPLE SPECIFICATIONS ................................................................................................... 7
SERVICE REQUEST AND SAMPLE SUBMISSION ................................................................. 8
SERVICE REQUEST FORM AND SAMPLE SUBMISSION (CREATING A NEW REQUEST) ...................................... 8
SERVICE REQUEST FORM AND SAMPLE SUBMISSION (EXISTING REQUEST) ............................................... 8
PREPARING SAMPLES FOR SHIPMENT ............................................................................................ 9
SHIPPING ADDRESSES FOR SAMPLES ............................................................................................ 9
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 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 nuclease-free water or in 10 mM Tris-HCl pH 7.58.5 (no EDTA should be added as its presence 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,

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.
Sample identification of the container(s) carrying the sample(s) must correspond exactly to what is
specified in the Request Form.
3
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 6 µL is used to first assess DNA Quality Control.
Table 1. Summary of DNA sample requirements
Source of
nucleic acid
gDNA
ChIP DNA
Required
Quantity
(µg)
Required
Volume
(µL)
Short insert
shotgun (TruSeq)
≥1.0
40 to 75
Short insert
shotgun (Nextera)
≥0.25
40 to 75
WGBS
≥2.0
40 to 75
3-10 kb Mate Pair
≥10
40 to 75
Target Capture
(SureSelect)
≥3.0
40 to 75
Target Capture
(Nextera)
≥0.25
40 to 75
Target Capture
(Roche Nimblegen)
≥1.0
40 to 75
Methyl Capture
(Roche Nimblegen)
≥2.0
40 to 75
ChIP-Seq
≥0.025
25 to 50
≥0.075
25 to 75
Library Type
Sequencing-ready Library¶

¶
Fragment size range of ChIP DNA or amplicons: 100-500 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
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 either returned to the
expeditor or will be re-plated in the proper plates. There will be additional fees for shipping back
samples or re-plating them.
If samples from multiple projects are submitted at once, they have to be on different plates.
All plates must be clearly labeled with the quote number, name of PI, and date of shipment.
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.
5
Guidelines for RNA Samples
Important General Considerations
When working with biological samples that are destined to be submitted for RNA profiling (e.g. RNASeq, 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.
All correspondence regarding completed or ongoing services must make reference to the
corresponding quote number (begins with “SCI…”), or to the corresponding Nanuq Project Name.
6
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.
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 Illumina libraries from
RNA
Application
Amount
(ng)
Minimal
concentration
(ng/µL)
Volume
(µL)
RIN*
Eukaryotic gene expression
(protein coding transcripts)
700
50
~ 10-15
>6.5
Prokaryotic gene expression
2000
100
~ 20-25
>6.5
Meta-transcriptomics
(multiple species)
2000
100
~ 20-25
>6.5
Coding and long non-coding
transcripts profiling
1200
75
~ 15-20
>6.5
Small RNA profiling (<200 nt)
2200
200
~ 10-15
>6.5
*RIN = RNA Integrity Number; assessed on a Bioanalyzer (Agilent)
7
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.
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.
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.
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
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, 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 box.
It is recommended to e-mail 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 samples:
Attn: MPS Services c/o Sylvie LaBoissière
McGill University and Génome Québec Innovation Centre, Suite 5300
740, Dr.-Penfield Avenue
Montréal (Québec) Canada
H3A 0G1
RNA samples:
Attn: MPS Services c/o Sylvie LaBoissière
McGill University and Génome Québec Innovation Centre, Suite 6300
740, Dr.-Penfield Avenue
Montréal (Québec) Canada
H3A 0G1
Ready-to-sequence libraries:
Attn: MPS Services c/o Alfredo Staffa
McGill University and Génome Québec Innovation Centre, Suite 5400
740, Dr.-Penfield Avenue
Montréal (Québec) Canada
H3A 0G1
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