Quality Control

All nucleic acids received by the GSL are subject to a QC/QA procedure that is designed to identify possible low-performing samples before they are used in protocols. If samples do not meet minimum standards, replacement samples can be submitted with new sample IDs. The GSL will not pool samples for any reason.

DNA Quality Control

DNA QC includes:

• quantification using pico green chemistry or Qubit
• visualization of DNA size on an agarose gel to check for degradation

Nanodrop concentration determination routinely over-estimates the amount of DNA in a sample since it is not dsDNA-specific and can be thrown off by contaminants. Therefore, if using nanodrop for quantification of your samples, be sure to aim for the top range of material requested as it is likely your total amounts will be 30-50% lower than expected.

Agilent 2100 electropherograms for two high-quality RNA samples. The top image is human and received a RIN of 10.0, while the bottom sample is non-mammalian, and the unexpected size of rRNA molecules caused scoring to fail.

RNA Quality Control

RNA QC includes:

• quantification via Qubit
• bioanalysis using the Agilent 2100 Bioanalyzer

In bioanalysis, the electropherogram of a total RNA sample is analyzed and assigned an RNA Integrity Number, or RIN. The highest quality RNA will have RIN scores of 10, with samples having RINs of 7.0 or greater generally performing well in most protocols. Non-mammalian species are often assigned lower RIN numbers due to variation in expected ribosomal RNA sizes.

Library Quality Control

Electropherograms for two libraries prepared by GSL clients. The top library has the expected profile, with a single peak of fragment sizes. The bottom library is likely overamplified, with a secondary peak around twice the size of the expected fragments.

Library QC includes:

• quantification via Qubit
• bioanalysis using the Agilent 2100 Bioanalyzer to check library peak size and for the presence of multiple peaks
• Kapa RT-PCR on a 10nM dilution to independently validate concentration and determine final concentration for sequencing

Using the above protocol, the GSL makes every effort to accurately determine library concentration and the appropriate sequencing dilution. However, for libraries created by other labs, there is sometimes difficulty in predicting the appropriate dilution, especially if the libraries have multiple peaks or consist of pooled samples. The GSL will document the quantitation process for the libraries in question and the final decision on sequencing concentration will rest with the user. The GSL cannot guarantee performance or sequencing output for libraries generated by outside users; therefore payment is required prior to sequencing.