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. 1 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 2 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, 3 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). 4 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. 5 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 6 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. 7 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. 8 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. 9 Appendix 1 10 Appendix 2 Lab 3 entrance Lab 3 entrance 11