Nucleic Acid Quality Considerations
The success of obtaining DNA sequence data from nucleic acid samples depends upon the quality of the nucleic acid submitted and the quantity of sample added to each reaction. We can accurately determine and control sample quantities but nucleic acid quality is the responsibility of service users. If users are not able to meet our quality specifications, we do offer extraction services for both DNA and RNA samples (please inquire). If you have additional questions or concerns, please contact us via the “Conversations” link under the User menu (top of the page).
Sample specifications depend upon the service requested.
All nucleic acid samples are quantified using a fluorescent, intercalating dye when they arrive in the laboratory. You will be notified if > 10% of samples do not meet our minimum concentration requirements. Concentrations do not need to be standardized across all samples but large scale adjustments to sample concentration (dilution or concentration) in our lab will incur additional processing fees.
1. Whole genome sequencing or “skim” genome sequencing
"TruSeq-equivalent" WGS library construction requires high molecular weight DNA that is free of RNA and other impurities. We recommend column-based DNA purification. DNA samples extracted with detergents (SDS or CTAB) with no further purification or samples that contain large amounts of RNA do not perform consistently. Treat samples with RNase prior to column clean-up. PCR-free libraries require 100 ul of DNA at a concentration of at least 25ng/ul. Please do not dilute your DNAs, as we will remeasure their concentrations. If PCR is performed during library construction then lower concentrations can be used. DNA samples may be shipped at ambient temperature.
Our 1/3 concentration Nextera skim sequencing library preps can work with far less DNA, at lower concentration. For these preps, we require a minimum volume of 10ul and a minimum concentration of 2ng/ul. We will re-quantify and dilute your submitted samples, so please submit them at a higher concentration (but <100ng/ul) if possible.
Similarly, for our Nextera XT library preps (standard or 1/5th volume) we require at least 15ul of DNA at a concentration between 10 and 100 ng/ul.
Required sample quality control (QC) data include a gel image or capillary traces of 10% of the DNA samples submitted (e.g., 8 to 10 representative samples from a 96 well plate). If running a gel, use 0.8-1% agarose stained with ethidium bromide. If possible, load at least 100 ng DNA and at least one size standard (500ng of a λ HindIII mass standard is recommended). Please label your samples and size standards clearly.
2. 3’RNA-seq
Samples should comprise un-degraded total RNA (at least 15ul, preferably 30ul) at a minimum concentration of 100 ng/uL. RNA extraction protocols should include a DNase treatment step followed by column-based purification. Do not heat RNAs to deactivate DNase. Samples should be shipped as ethanol precipitates, dried or frozen on dry ice. If shipped frozen, thawed samples will not be accepted for processing.
You will be required to submit a gel image or capillary trace for ~10% of the RNA samples you submit (e.g., 8 to 10 representative samples from a 96 well plate). Please label your samples and size standards clearly.
3. rAmpSeq for maize or cassava
Because rAmpSeq is a PCR-based genotyping protocol, lower quality DNA (degraded or low concentration) is acceptable. We recommend using a cheap DNA extraction method such as SDS- or CTAB-based protocols. These samples may be shipped at ambient temperature and gel images or other QC data are not required. If a sodium hydroxide-based extraction protocol is used (e.g. Extract-N-Amp) samples should be shipped as ethanol precipitates or dried.