Sample Quality
Sample quality is the single most important factor in the success of a sequencing performance!
- Keck Genomics Platform (KGP) accepts samples as either tissue, isolated nucleic acids, or completed libraries. Although KGP will accept tissues and can perform isolation of nucleic acids, we cannot guarantee performance. The customer will assume all risk of down-stream performance of any samples that are below recommended quality.
- Samples submitted to KGP should:
- Contain no sensitive patient identifying data that violates HIPPA.
- Have proper consents in place.
- Have all necessary service approvals and agreements in place.
- Have appropriate sample identification.
- Be submitted with a manifest containing a list of submitted samples as well as sample identifiers.
- Be safe and non-hazardous.
- All Samples must meet KGP’s Requirements, otherwise customer will assume all risks:
- KGP performs thorough quality control using the Agilent 4200 TapeStation unit, Qubit, or both before each application. Therefore, it is critical to provide enough volume to allow for quality control testing. An additional volume 3-5 μL (varies with concentration) should be utilized in quality control assessments. KGP is not responsible for any adverse outcomes of downstream performance contributed to lack of sample volume and concentration as it is the responsibility of the sample submitter to ensure there is adequate volume in sample submissions to account for quality control assessments.
Low-Input or Low-Quality Samples (FFPE)
- Samples that contain either lower than recommended concentration or quality may impact overall quality of the final library. KGP cannot guarantee performance of any sample that is below the recommended quality and thus will flag any such sample for a discussion with the customer prior to pooling for sequencing.
- Low-quality libraries are very unpredictable, and – in many cases – the true impact can only be revealed in poor performance of the final sequencing data
- Poor performance of generated libraries can be a result of high duplication rates, variation of insert size, poor probe hybridization, and decreased complexities contributed by the unpredictable nature of lower quality samples.
- Customers will assume any risk of the overall performance of FFPE and lower quality samples.
- Due to the overall poor-quality nature of FFPE samples, KGP has developed workflows designed to maximize the success when working with such samples; however, we cannot guarantee the performance of library preparation or final data quality, as vast numbers of samples are considered poor-quality and will not pass our standard quality control. KGP understands the value of subpar FFPE samples and will continue pushing the boundaries in developing better protocols to maximize the quality of data, but the customer must assume the risks involved and provide payment for services regardless of outcome when working with samples that do not meet KGP’s QC specifications.
- The general rule of thumb when considering downstream assays for FFPE (DNA) samples is that FFPE tends to generate better WES (Whole-Exome Sequencing) data than WGS (Whole-Genome Sequencing) data.
- Additionally, FFPE (RNA) samples are too degraded for a polyA selection and need to follow the ribosomal degradation protocol. Generally, when working with highly degraded poor-quality samples, increase in overall quantity can occasionally compensate for poor-quality that may influence better results.
- FFPE (RNA) performs best with a DV200 above 30%. KGP is not responsible for the outcome of the samples if the submitted samples are below this standard. KGP will notify the customer if any of the samples submitted fall below this value. Customer approval must be obtained to proceed in such instances. If KGP is given approval to proceed with library preparations when DV200s fall below 30%, KGP is not responsible for any poor outcomes as it is the responsibility of the customer to assume all risks once they have been notified and given the opportunity to either cancel library preparation, resubmit samples, or proceed with library preparation.
- In instances to which FFPE (RNA) samples are shown to have a history of poor quality, KGP may suggest that samples skip the QC process.
- KGP must notify and gain permission from the client to proceed with library prep without performing a QC check.
- If the client agrees to not perform a QC check on their submitted samples, the client understands that KGP is not responsible for the library prep or sequencing outcomes.
Sample Storage and Submission Guidelines
Please contact us prior to isolation and sample preparation, as any sample that is submitted that does not meet the requirements will be subjected to a $30/sample handling fee.
Sample Types
- Blood Samples
- Blood should be stored in lavender tubes (EDTA-coated), Trizol, or PAXgene tubes.
- Blood should never be stored in heparin-tubes as heparin is a potent amplification inhibitor and can affect downstream process and analysis results.
- Lavender tubes (EDTA-coated) should follow be stored in proper storage conditions of 4°C for no longer than 3 days. For long-term storage, it is recommended to keep tubes at -80°C.
- KGP recommends PAXgene tubes as the standard for blood sample submission. Blood collected in PAXgene tubes remains stable under various conditions, depending on the specific tube and the intended sequencing assay. For proper storage, please consult the specified guidelines. For long-term storage, we recommend keeping PAXgene tubes at -80°C to maintain sample quality.
- All shipped blood samples need to have appropriate shipping labels and be shipped on dry ice to ensure quality.
- Nucleic Acid Samples
- Nucleic acids should be stored in buffers such as 10mM Tris-HCl, laboratory grade ddWater, or Elution Buffer with concentration of EDTA not to exceed 0.1mM.
- KGP accepts samples in 5 mL, 2.0 mL, or 1.5 mL tubes for storage. However, we prefer samples submitted in 96-well full-skirt plates that can withstand -80°C temperatures. Regardless of container, all samples should be properly sealed to endure shipping and storage at -80°C.
- A $30 per sample handling fee will be charged for submissions exceeding 16 samples in 5 mL, 2.0 mL, or 1.5 mL tubes.
- Sample submissions of RNA should be sent on dry-ice and overnight. KGP is not responsible for poor performance if samples were received with little to no dry ice.
- For thorough quality control, downstream processing, and potential repeats, we recommend samples with:
- A minimum volume of 15-20 μL
- A minimum concentration of 4 ng/μL
The customer is responsible to provide KGP with proof of BUA or IRB that is associated with every sample that is submitted.
To avoid weekend deliveries, submit samples between Monday and Wednesday, and allow extra business days around holidays.
Sample Submission Address:
Attention: Keck Genomics Platform
Keck School of Medicine of University of Southern California
1450 Biggy Street, NRT 2501
Los Angeles, CA 90033
Tel:(323) 442–0288
Sample Policies
- All samples that do not meet quality requirements may be submitted for sequencing by customer’s written consent, but KGP cannot guarantee quality of the data and all the expenses will be the responsibility of the customer.
- Any sample that is below recommended quality will be treated as a low-quality sample. The customer will have the opportunity to provide KGP with a brand-new sample with handling fees applied. The new sample will be assigned a new ID and will be treated as a new sample.
- At the customer’s request, any unused samples can be returned to the customer with applicable shipping and handling fees.
- Any sample that is mislabeled or does not meet KGP’s submission guidelines will be subject to a $30/sample fee.
- For 16 or more samples, please use full-skirt PCR plates. Other formats may incur a $30/sample handling fee.
- The customer is responsible for performance and full cost of the sequencing services performed on submitted libraries.
- The customer will be notified by email upon receiving of samples.
Quality Control (QC)
- Nucleic Acids Quality Control
- DNA
- Qubit, Bioanalyzer, TapeStation are recommended standards
- DIN > 5
- Nanodrop, although informative is not recommended
- RNA
- Qubit, Bioanalyzer, Tapestation are recommended
- RIN (RNA integrity Number) > 7
- On FFPE we also calculate DV200 and follow Illumina’s guidelines of > 30% to be considered into library preparation.
- Library
- Qubit, Bioanalyzer, Tapestation are recommended
- Kapa qRT-PCR on a 5nM dilution is recommended to determine the final concentration
- KGP cannot guarantee performance of libraries that are not generated in-house and therefore an advance payment is required prior to the initiation of the sequencing run.
- DNA
Sequencing
- KGP will perform sequencing on all samples as agreed by the customer’s quote.
- Sequencing is to be performed within 6 weeks after arrival of all samples and each sample either passes quality control or KGP has given consent by customer to proceed.
- The customer will not be charged additional cost for any added sequencing depth.
- Any sample that has at least 85% or greater target coverage is considered a success and will not be subject to re-sequencing.
- It is important to note that inclusion of a poor-performing sample may not only result in poor performance of the sample itself, but may also impact the entire run and as a result, affect target coverage across all samples. KGP cannot guarantee agreed performance of the entire run if customer desires not to exclude a flagged low-quality library.
Data and Informatics
- Deliverables of sequencing data are raw reads .fastq files, aligned to a genome .bam files, in case of DNA copy number files, variant files .vcf, for RNA expression files, and general stats file. Any analysis beyond basic informatics becomes collaborative in nature with a possibility of additional fees that may apply.
- Any data stored on our server that is older than 3 months will be subject to deletion without notification.
- Data will be distributed through Globus or via external drive per customer request. KGP will only be responsible for copying the data to single location and any additional data movement will be subject to additional fees. Due to increased risk of malicious codes, KGP will not accept external drives provided by customer.
Payment
- Keck Genomics Platform requires proof of account prior to the release of any data.
- Customer will only be invoiced for services that were actually performed.
- If customer decides not to sequence a poor-quality sample, the customer will only be charged $30/sample for sample handling.
For payment details please contact:
Kendra L. Bergen
Budget/Business Analyst
(323) 442-7823
kendra.bergen@med.usc.edu
Ashley Jauregui
Research Laboratory Technician III/ Supervisor of Laboratory Operations
(323) 442-0859
ashleyj2@usc.edu