Sample Submission Guidelines

Sample Submission Guidelines
As part of our continual quality improvement program we are making changes to our
sample submission process. The appropriate DNA/RNA concentration, storage
conditions and ways to deliver your sample(s) are presented below. Timely
completion of your project depends on you following these guidelines.
We expect that your samples will be supplied in the correct containers and at the
required concentration and quality (see details below). With the high volume of
samples we receive on a daily basis, QC is carried out in batches of 96.
To prevent delays to projects that are batch-processed using our automated
systems, in the event that a sample does not meet our QC criteria, we will still
proceed with the library preparation. Your project manager will contact you to
inform you of the problems with sample QC. It will then be at your discretion
whether you sequence the library. If you choose not to sequence the library and
instead provide us with a replacement sample(s), drop that sample or cancel your
project you will still be charged for library prep and sample QC. Additional costs for
QC and library preparation will be levied for each replacement sample(s). All new
samples will be added to the end of the queue for the next batch of samples to be
processed.
THIS DOCUMENT CONTAINS IMPORTANT INFORMATION ABOUT
QUALITY CONTROL AND SUBMISSION OF YOUR SAMPLES. PLEASE READ
IT CAREFULLY PRIOR TO SENDING SAMPLES.
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Sample Submission Overview
The schematic below is designed to give you an overview of the steps that you are
required to take to ensure safe delivery of your samples.
QC samples
< 8 samples in tubes, ≥8
samples in plate
Send signed quote, PO and
sample submission form. Wait
for confirmation
Package Appropriately at the
Correct Temperature
Deliver samples to WTCHG
Ship samples to WTCHG
Relax
Sample QC
DNA…………………………………………………………………………………………………………….3
PCR Amplicons……………………………………………….……………………………………………3
ChIP…………………………………………………………………………………………………………….4
RNA…………………………………………………………………………………………………………….4
Pre-prepared Libraries….…………………………………………………………………………….5
Packaging Samples………………………………………………………………………………………………...7
Submitting Samples for those with access to WTCHG………………………………………….…8
Submitting Samples for those without access to WTCHG ……………………………………...9
Appendix 1……………………………………………………………………………………………………………..10
Appendix 2……………………………………………………………………………………………………………..11
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Sample QC
We recommend taking the following steps for the different sample types to ensure
that they meet our QC criteria. If you are unsure please contact your project
manager prior to sending the samples. If your sample(s) do not meet our QC criteria
we will still proceed to make a library. A sample of poor quality is likely to produce a
poor, or biased, library. The cost of library prep and sample QC will be applied for all
primary libraries, with an additional cost levied for any replacement samples.
Replacement samples will be added to the end of the queue, so increasing
turnaround times.
DNA
We require 1-5μg of DNA normalized to a concentration of 50ng/μl in 10mM Tris-Cl,
pH 8.5 (please note, for SureSelect captures, we require 3-5μg). Quantification
should be done by Qubit or Picogreen. Nanodrop overestimates the amount of
material present and reliance on this method of quantification will risk the library
failing. Nanodrop should be used to confirm that the 260/280 ratio is between 1.8
and 2 and that the 260/230 ratio is between 2-2.2. Material should be run on a 0.7%
agarose gel. Samples should give distinct bands with no smearing, as shown in the
figure below. Samples should be RNase treated.
Figure 1: Good quality DNA, clear, crisp bands, no smearing
PCR Amplicons (final product for library prep)
PCR amplicons should either be longer than 1.5Kb or if smaller should be
concatenated (Druley TE, Vallania FLM, Wegner DJ, Varley KE, Knowles OL, et al.
(2009) Quantification of rare allelic variants from pooled genomic DNA. Nat Meth 6:
263-265).
Each PCR product should be cleaned up using a PCR purification kit (e.g. the Qiaquick
kit from Qiagen) or Ampure XP beads (Beckman Coulter Genomics). All amplicons
should be run individually on an agarose gel or on a bioanalyzer chip. Only a single,
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clean product should be obtained. Another
agarose gel should be run after concatenation to confirm that a high molecular
weight smear is present.
Where amplicons are being pooled prior to delivery to HTG for library preparation,
this must be done so that each amplicon is represented in equimolar concentrations
within the pool. The size of each amplicon within the pool should be stated in the
“notes/description” column of the submission form.
We require 1-5μg of DNA normalized to a concentration of 10-50ng/μl in no more
than 100μl of 10mM Tris-Cl, pH 8.5. Quantification should be done by Qubit or
Picogreen. Nanodrop overestimates the amount of material present and reliance on
this method of quantification will risk the library failing. Nanodrop can be used to
confirm that the 260/280 ratio is between 1.8 and 2 and that the 260/230 ratio
between 2-2.2.
ChIP
We require 10ng of material. Quantification should be done by Qubit or picogreen.
Samples should be in a volume of 45μl or less of ultrapure water, only 40μl will be
used for the library preparation. Samples should be run on a High Sensitivity
bioanalyzer chip to test the fragmentation. For the library prep, we require that
there is material in the 50-350bp range. We recommend that a control real-time PCR
is run to confirm that the expected enrichment is present prior to submitting the
sample for sequencing. Although there is no defined limit for the amount of
enrichment that can be detected by sequencing, in some cases, 2-3 fold enrichment
by qPCR has not been detected adequately by sequencing.
RNA
It is recommended that all RNA samples are DNase treated with a PCR-grade DNase.
The enzyme should be inactivated with EDTA and heat after treatment and then
cleaned up. Ensure that RNase-free tubes, plates, lids and tips are used. Take all
additional precautions suitable for RNA work.
We require 1-6μg made up to 30μl in ultrapure water (for small RNA library preps we
need 3μg of total RNA in 7μl of ultrapure water). Quantification should be done
using the Qubit RNA Broad-Range Assay kit. Nanodrop is a viable alternative to give a
rough quantification of RNA samples but please be aware that it tends to
overestimate. Samples should be run on a RNA bioanalyzer chip to determine the
quality, RIN values should be 8 or above. If that is not possible an estimate of
integrity can be obtained using a 1% RNA gel, below is an example of good quality
RNA (Figure 2).
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Figure 2: Lanes 1-3 are nuclear RNA, lanes 4-6 are cytoplasmic RNA (figure taken
from http://www.norgenbiotek.com).
Pre-prepared Libraries
When submitting prepared libraries, these must be at least 10nM, when quantified
using fluorescence (see the table below for a guide to the concentrations required).
We require at least 15L of each prepared library.
Minimum Concentration of DNA
(ng/L)
1.7
2
2.6
3.3
Average Size of Library (bp)
250
300
400
500
N.B. When optimal library concentrations cannot be obtained it may still be
possible to sequence the library but the amount of data cannot be guaranteed,
please discuss this with your project manager.
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Summary Table
Type of
Sample
DNA or
amplicons
DNA for
SureSelect
capture
Amount
required
Concentration Volume
OD
260/280
Agarose
gel
1- 5μg
10-50ng/μl
100 μl
1.8 -2.0
0.7%
3-5 μg
30-50ng/μl
100 μl
1.8 -2.0
0.7%
mRNA
1-6μg
35-200ng/μl
30 μl
~2
Small RNA
3μg
200ng/μl
7 μl
~2
ChIP
10ng
>0.25ng/μl
45 μl
Prepared
Libraries
~45ng
~3ng/μl
15μl
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Bioanalyzer
RNA 6000
Nano
RNA 6000
Nano
High
Sensitivity
Chip
DNA 1000
or High
Sensitivity
Chip
Packaging Samples
The following guidelines are to ensure that your samples reach us in the same
condition as you sent them.
1. Samples should be clearly labeled and labels should match those given on the
submission form. An example of a correctly completed submission form can
be seen in Appendix 1.
2. If you have fewer than 8 samples, they should be submitted in 1.5ml
microcentrifuge tubes, we recommend Eppendorf LoBind tubes. If you have 8
or more samples, they should be in a fully skirted 96-well plate
(ThermoFisher Thermo-Fast 96 Skirted plates, catalogue #AB-0800). Samples
that are to be multiplexed should be grouped and assembled on a plate so
that samples in a given multiplex are in consecutive wells (overall plate layout
should be in columns). If you are multiplexing different sample types, please
discuss this with your project manager.
3. If the samples are in the wrong tubes, we reserve the right to return the
samples at your cost or to charge a processing fee.
4. Tubes should be packaged together in a sealed container, plates should be
placed within a plastic bag. The sealed container or sample plate should be
labeled with your name and quote number.
5. Only one aliquot of each sample should be submitted unless by prior
arrangement.
6. All RNA samples should be on dry ice.
7. All DNA samples or libraries should be on dry ice if being shipped or if to be
left in WTCHG reception area.
8. Please email your project manager with the submission form at least 2 days
in advance of bringing or sending your samples and wait for confirmation
that your paperwork has been completed.
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Submitting Samples for those with Access to WTCHG
When dropping off your samples
1. There is a designated freezer for sample drop off. The freezer is located
within Room 10/086, which can be accessed from the south side of Lab 3. A
plan of Lab 3 and the location of the freezer can be seen in Appendix 2.
2. Samples should be left in this dedicated area between 10am and 2pm on
standard working days. This freezer will not be checked other than between
these times so we recommend only to leave samples between 10am and
2pm in order that your samples are not lost or defrosted if the freezer is
opened or fails after this time.
3. The labeled plate or box/bag of tubes should be placed in appropriate
drawer; either the one for DNA or the one for pre-prepared libraries.
4. On top of the freezer there is an area in which to leave RNA samples on dry
ice.
5. There are also two folders on top of the freezer. Your sample submission
form will be in the “pending” folder. Please sign this and transfer it to the
“received” folder.
6. After 2pm, nobody will return to check for samples until the following
working day.
7. If you are unable to drop off your samples between 10am and 2pm, please
arrange for a colleague to do so or wait until the following working day.
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Submitting Samples for those without Access to WTCHG
1. Samples should only be shipped between Monday and Wednesday (or 2
working days before the start of the weekend in the case of bank holidays).
2. All samples should be on dry ice. Some couriers have specific guidelines for
shipping samples on dry ice and should be contacted for details prior to
packaging up your samples.
3. We recommend that you ship the samples to us. Please send to:
High-Throughput Genomics (Sequencing),
Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford.
OX3 7BN
4. If it is not possible to ship samples, it will be possible to leave samples in a
cupboard in reception (see image below) between 10am and 12pm- this area
is not secure and will become warm, please put all samples on dry ice.
5. After 12pm, nobody will return to check for samples until the following
working day.
6. If you are unable to ship or drop off your samples in this time slot, please
wait until the next working day or ask a colleague to send the samples in your
absence.
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Appendix 1
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Appendix 2
Lab 3 entrance
Lab 3 entrance
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