Next Generation Sequencing and Bioinformatics
Illumina sequencing - Sample submission
We strongly recommend that you send us your quality control data before submitting your samples to the GenePool. Our technologists will be happy to advise you on the suitability of your samples for Illumina sequencing. Please also note that samples delivered to us without QC data will be returned for quality control by you.
We will assess the suitability of your samples upon receipt. However, please note that we cannot guarantee that your sample will perform well in downstream procedures even if it passes quality control criteria. Should one of your samples pass our quality control but fail to generate suitable data, we will ask you for a replacement and repeat the procedure at no cost to you.
Minimum input and concentration requirements
All submitted samples must meet the requirements below to pass our quality control (minimum requirements apply to one sequencig library). Please contact us to discuss the suitability of your samples if you are unable to provide the quantity and/or concentrations specified below.
1 For mate-pair libraries, we require at least 20 µg of high molecular weight genomic DNA depending on the virtual insert size. Please contact us to discuss the specific requirements of your project
2 Please supply PCR products and sequencing amplicons (i.e. PCR products incorporating the Illumina adapters) as gel, column or AMPure XP-purified fragments
We require DNA that is free of RNA (i.e. RNAse treated, we recommend Epicentre RiboShredder), minimally degraded, and free of other contaminants. Note that if you carry out an RNAse treatment, samples must be purified using a suitable method (e.g. phenol-chloroform) before submission. To ensure that your samples make quality libraries, we also recommend that you check that your DNA is free from inhibitory compounds by performing a test restriction digestion (please contact us if you need advice on setting up a such a test).
We request that you provide the following QC data for each DNA sample on the sample submission form:
Important note. We find that DNA concentration as determined by spectrophotometer (e.g. NanoDrop) is highly unreliable. If you are unable to determine concentration by fluorometry or gel electrophoresis then please send us three times the minimim amount and be prepared that we may ask for more sample.
To obtain the best quality RNA we recommend a phenol-based extraction followed by DNAse treatment (Turbo DNase, RQ1 DNase) then solid-phase purification (RNeasy, Nucleospin, or MEGAclear).
We request that you provide the following QC data for each RNA sample on the sample submission form:
Important note. We find that RNA concentration as determined by spectrophotometer (e.g. NanoDrop) is highly unreliable. If you do not have access to a fluorometer or a Bioanalyzer, please send us three times the minimum amount and be prepared that we may ask for more sample.
For further details on assessing RNA quality, you may wish to read the following publications:
Please submit Illumina libraries ready for sequencing that have been purified with a suitable PCR cleanup kit. Quantification can be done by NanoDrop spectrophotometer, but fluorometry (e.g., Qubit, Invitrogen) is preferred. Please also include either a gel picture or, preferably, a Bioanalyzer trace.
We will check your library before processing and contact you if we think there are any issues. Unfortunately we cannot offer a guaranteed yield or quality for libraries prepared outside of the GenePool but there are QC steps that you can take to minimise the risk of a poor sequencing run. For example, you can quantify your library by using a molecular weight ladder with known concentrations of fragments similar to the size of your full-length product (Fermentas, for example, has a good selection). Use several microlitres of the purified product and run on 10% PAGE or a high percentage agarose gel (>1.8%). You should stain the gel after running for accurate quantification (ethidium bromide works well but for maximum sensitivity we recommend using SYBR Gold). In addition, we strongly recommend that you check your library by cloning and Sanger sequencing before proceeding with sample submission.
|The GenePool has core support from:||GenePool admin site|
|The University of Edinburgh
School of Biological Sciences
The Darwin Trust