UWBC Illumina Sequencing Policies

Spring 2018

Please thoroughly review these policies prior to submitting samples. If you have any questions regarding this policy or any other aspect of next generation sequencing, please contact us at nextgen-seq@biotech.wisc.edu.

Sample and Run Queue

Projects will not be entered into the Illumina run queue until samples and completed submission form are in the possession of the DNA Sequencing Facility. A flow cell will not be run until all lanes are filled. Upon request, UWBC will give you a tentative run date. We strive to honor this run date; however, there are issues beyond our control that may delay your run. In the event of a delay greater than a week, UWBC will email users to inform them of an updated run date. In order to expedite sequencing, please feel free to notate different acceptable run types. Typical turnaround time is 6 weeks.

Minimum and Optimal Sample Requirements

If the following initial QC requirements are not met, you will be informed via email. You may elect to either resubmit samples of sufficient quality and quantity or proceed with the existing samples. Should you choose to proceed with samples that do not meet our requirements, the samples will be run AS IS, and we make no assurances on the quality of downstream data.

User Responsibilities

  • Dropped off samples must be prepared to UWBC specifications. Submission guidelines can be found on the DNA Sequencing webpage.
  • The organism name and a reference genome of a phylogenetically analogous organism (e.g. hg19 for Homo sapiens) must be supplied, if available.

Options for resubmission

If concentrations are too low:

  • Additional sample material can be submitted. Additional quantity/quality measures of this material will be done at the submitting lab’s expense (price determined using current rates for number of samples requiring measurement).
  • The entire sample submission can be returned to the submitting lab. The lab is encouraged to precipitate or purify the DNA and elute in an appropriate volume. Vacuum centrifugation is discouraged as it will also concentrate inhibitors.

If concentrations are too high:

  • The entire sample submission can be returned to the submitting lab and the sample(s) in question can be diluted appropriately from the measurements provided by the UWBC.
  • The staff at the UWBC can perform sample dilution at submitting lab’s expense (price determined using current rates for number of samples requiring measurement).

UWBC Deliverables (approximation* per lane)

The UWBC strives to deliver data meeting or exceeding the following metrics:

  • MiSeq v2: 12M reads passing filter, 75% bases Q30 (99.9% accuracy) or greater
  • MiSeq v3: 15M reads passing filter, 70% bases Q30 (99.9% accuracy) or greater
  • MiSeq nano: 800,000 reads passing filter, 75% bases Q30 (99.9% accuracy) or greater
  • MiSeq micro: 4M reads passing filter, 75% bases Q30 (99.9% accuracy) or greater
  • HiSeq2500 (High Output): 200-220M reads passing filter, 75% bases Q30 (99.9% accuracy) or greater
  • HiSeq2500 (Rapid): 140M reads passing filter, 80% bases Q30 (99.9% accuracy) or greater
  • Shared MiSeq 2x300 (Amplicon libraries): 20,000 reads per submitted sample
  • FASTQC and Run Summary Report will be delivered for each library. These files provide overviews of run quality. Reads will not be trimmed for any reason.
  • Data are delivered in FASTQ format within 5 business days of run completion. Further analysis may be contracted separately with the Bioinformatics Resource Center (BRC) at brc@biotech.wisc.edu.

The UWBC strives to ensure all Facility prepared libraries meet these specifications. As noted*, run success is DNA and genome dependent to a certain degree so variation occurs and some libraries will not perform as well as expected. Facility prepared libraries that grossly underperform due to Facility or instrument errors will be re-run at the earliest possible date; however, there may be a wait for a run that will meet or exceed the requested parameters.

Customer prepared libraries that do not meet these specifications can be re-run at the submitting lab’s expense.

* Approximation because there are many different factors: species, kit used, phiX %, GC content, etc. Please contact nextgen-seq@biotech.wisc.edu to discuss concerns.

Exceptions

While we will make every effort to create successful sequencing runs with high quality data output, we cannot be responsible for improperly prepared samples. Therefore, we make no guarantees on the quantity and quality of data generated from Illumina sequencing if:

  1. Libraries are prepared by the user, where we have minimal control over quality.
  2. Custom primers are supplied with incorrect sequences or inappropriate melt temperatures
  3. Samples are submitted that do not meet UWBC minimum submission requirements (stated on guidelines located on UWBC website).
  4. Non-Illumina barcode/index methods are used. Barcodes placed at the 5’end of the sequence and those with no redundancy are especially prone to read errors.

If your sequencing libraries grossly underperform, or if you have other questions/concerns regarding your data, please contact us immediately at nextgen-seq@biotech.wisc.edu to address your concerns.

Data Storage

Deliverable sequence data will be stored in your download directory. These files will be kept for a limited time by the DNA Sequencing Facility but the PI is ultimately responsible for long term storage. We store the raw bcl file output from each sequencing run for approximately 30 days. If you wish to have a copy of this data, you must purchase the entire run, provide a clean external drive, and notify the DNA Sequencing Facility prior to the beginning of your sequencing run.

Sample Storage

All input DNA submitted after 4/23/18 will be stored for 6 months and then disposed of.
All customer prepared libraries submitted after 4/23/18 will be stored for 6 months and then disposed of.