BioMicroCenter:Sequencing Quality Control: Difference between revisions
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== Possible QC Techniques == | == Possible QC Techniques == | ||
* [[BioMicroCenter:Nanodrop | * [[BioMicroCenter:Nanodrop ND-1000|Nanodrop ND-1000]]- this method allows you to quantify samples without diluting them. It has a detection limit of about 2ng/ul to 3700ng/ul. However, the Nanodrop is not recommended for sequencing samples because this method is not specific to dsDNA and has proven to have great variation at lower concentrations. Use of the BMC Nanodrop is FREE | ||
* [[BioMicroCenter:2100BioAnalyzer|Bioanalyzer]] - This method quantifies samples sequentially by their size by molecular sieving and are then detected by fluorescence. This method is only suggested for samples suggested for samples exceeding 10ng/ul and has shown to be less reliable at lower concentrations. Available at the BMC for $10 per sample | * [[BioMicroCenter:2100BioAnalyzer|Bioanalyzer]] - This method quantifies samples sequentially by their size by molecular sieving and are then detected by fluorescence. This method is only suggested for samples suggested for samples exceeding 10ng/ul and has shown to be less reliable at lower concentrations. Available at the BMC for $10 per sample | ||
Revision as of 14:21, 12 January 2009
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Why is QC Important?
It is very important to supply us with a reliable measurement of the concentration and quantity of starting material so that we can prepare the sample for sequencing. If a sample is too concentrated the cluster density will be too high and the GA will not be able to detect them properly. If a sample is too dilute the cluster density will be too low and there will not be enough reads for the pipeline to make base calls. Having reliable QC measurements allows us to optimize the cluster density and the number of reads per lane and maximize the quality of data produced. QC can either be run before sample submission and specified upon testing request or can be run by the BMC. We strongly recommend that you allow us to run QC of samples.
Possible QC Techniques
- Nanodrop ND-1000- this method allows you to quantify samples without diluting them. It has a detection limit of about 2ng/ul to 3700ng/ul. However, the Nanodrop is not recommended for sequencing samples because this method is not specific to dsDNA and has proven to have great variation at lower concentrations. Use of the BMC Nanodrop is FREE
- Bioanalyzer - This method quantifies samples sequentially by their size by molecular sieving and are then detected by fluorescence. This method is only suggested for samples suggested for samples exceeding 10ng/ul and has shown to be less reliable at lower concentrations. Available at the BMC for $10 per sample
- Qubit - This method quantifies samples by using fluorescent dyes that are specific for dsDNA, RNA or protein. This method is used in the Fraenkel lab to quantify their samples for sequencing. Not yet tested at the BMC but information about this application can be found at http://openwetware.org/index.php?title=BioMicroCenter:Sequencing_Quality_Control&action=edit&redlink=1
- PicoGreen - This method makes use of a fluorescent dye, PicoGreen, that binds specifically to dsDNA. The fluorescence of this dye can by measured a few different ways, the manufacturer states that the Qubit can be used and the Boyer lab uses the photospectrometer, Typhoon, located in the Baker lab. This method has not been confirmed at the BMC yet. More information can be found at: http://probes.invitrogen.com/media/pis/mp07581.pdf
- RT-PCR, SYBERgreen assay - This assay allows for the amount of DNA that will actually bind to the flowcell to be quantified by making use of the sequencing primers in the QPCR assay. This method is currently being used at the Broad Institute and is coming soon to the BMC!!
