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. If a sample is too concentrated the cluster density will be too high and the GA will not be able to detect them properly, Illumina will soon be releasing a software update which should help with this. If a sample is too dilute the read count will be too low and could have a negative affect on the data produced. Having reliable QC 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, and provided 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. The NanoDrop has a detection limit of about 2ng/ul. However, the NanoDrop is not recommended for sequencing samples because it is not specific to dsDNA and has not shown good results with the Solexa sequencer.

• Bioanalyzer - This method separates samples via molecular sieving, detects them using fluresence, and quantisifes them sequentially by their size. The Bioanalyzer is only suggested for sequencing samples exceeding 10ng/ul and has shown to be less reliable at lower concentrations. Available at the BMC for $10 per sample.

• Qubit - TThis method quantifies samples by using fluorescent dyes that are specific for dsDNA, RNA or protein. The Qubit is used in the Fraenkel lab to quantify their samples for sequencing. It is 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 be measured in 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!!