BioMicroCenter:Sequencing Quality Control
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Why is QC Important?
It is very important to have a reliable measurement of the amount of starting material so that the sample can be prepared for hybridization and amplification on the flow cell. Samples must be at a final concentration of 10nM to be prepared to be run onto the flowcell, a 1.3ug/ul, 200bp sample is approximately 10nM.
Currently the optimum cluster range is from about 80,000 to 140,000. If a sample is too concentrated or if the fragment sizes are too variable the clusters will overlap and the GA will not be able to detect the reads properly. And if the sample is too dilute you will not get as many reads as possible from the lane. Illumina will soon be releasing a software update that will increase the optimum cluster density and allow for higher density reads to be better detected. Having reliable concentration measurements allows us to optimize the number of reads per lane and maximize the quality of data produced.
QC can either be run before sample submission or can be run by the BioMicro Center.
Possible QC Techniques
- NanoDrop ND-1000- The NanoDrop is one of the most commonly used tools to measure the concentration of DNA in solution. The NanoDrop has a detection limit of about 5ng/ul. Unfortunately, due to noise at the lower detection limit, samples speced on the Nanodrop have not shown reliable results on the Solexa sequencer. We do not recommend using the NanoDrop as the primary method of determining concentration for samples on the Sequencer.
- Bioanalyzer - The Bioanalyzer produces data similar to that of gel elctrophoresis. However, this technique requires much less sample input and provides quantification data. The bioanalyzer is currently the most commonly used tool for determining the concentration of samples for sequencing. However, the lower the concentration of the sample or the wider the distribution the higher the error. Due to this the Bioanalyzer is not recommended as the primary method of determining concentration for samples that are less than 20ng/ul. Available at the BioMicro Center for $10 per sample.
- Qubit - The Qubit is a selective flurometer that uses fluorescent dyes that are specific for dsDNA, RNA or protein to quantify samples. The Qubit is used in the Fraenkel lab to quantify their samples for Solexa sequencing. Information about this application can be found at: http://openwetware.org/index.php?title=BioMicroCenter:Sequencing_Quality_Control&action=edit&redlink=1
- PicoGreen - PicoGreen is a fluorescent dye that binds specifically to dsDNA and allows for quantification. PicoGreen can be measured in a few different ways, the Boyer lab uses the photospectrometer, Typhoon, located in the Baker lab and manufacturer states that the Qubit can be used as well. More information can be found at: http://probes.invitrogen.com/media/pis/mp07581.pdf
- RT-PCR, SYBERgreen assay - This assay uses primers that are specific for the adaptors used to create the library during sample preparation. Having this specificity allows for the amount of DNA that will actually bind to the flowcell to be quantified. The RT-PCR assay is recommended in addition to the techniques explained above and provides additional and more precise concentration information. This method is currently being used at the Broad Institute and is coming soon to the BioMicro Center.
