Frequently Asked Questions

What is Freedom Markers?

Freedom Markers specializes in tGBS® genotyping-by-sequencing (GBS) for plant species. Customers interested in obtaining quotes from us for plant species with genomes ≤5Gb in size are encouraged to use our online form found here. For other inquiries/questions not covered below, please contact us with your inquiry and our friendly staff will attempt to answer you as soon as possible.

How to Proceed:
  • Fill out & submit the online order form
    • Note: the address listed on the order form will be used to ship tissue collection supplies. This address will also be used to ship final results for those customers who have chosen the physical hard drive delivery option
  • Once the order form has been submitted, an e-mail confirming all project details with an official quote and instructions on how to initiate the project will be sent. This process should only take a few minutes, but please allow up to twelve hours to receive the quote e-mail. If you have not received a quote e-mail after twelve hours, please check your spam filter. If you still cannot find the quote e-mail, please contact us
  • Once you decide to initiate the project, submit a purchase order (PO) from your purchasing department using the URL provided with the quote
  • Customers who have chosen to send tissue samples will have the following supplies mailed out at no additional charge:
    • Collection plates, collection tubes, airpore tape for lyophilization (freeze drying), and strip caps for shipping
    • It is essential that all customers ship tissue samples in the provided plates. This ensures each individual sample will be properly and accurately tracked throughout the entire genotyping process
  • Collect samples in supplied tubes and lyophilize (if possible)
  • Ship samples as listed below
Sample Requirements:
  • See image for examples approximating how much tissue should be collected per well
    • ~30 mg of freeze-dried (lyophilized) tissue is required per sample
    • Tissues should be young leaves with a bare minimum amount of fibrous material (stems, roots, mid-ribs, etc). Samples consisting a *significant* amount of fibrous material can cause issues with DNA extraction and will likely lead to poor quality sequencing results
  • If either excessive or inadequate amounts of tissue samples have been received, it will not be possible to extract DNA and the plates will be discarded. Customers will be asked to send replacement samples
  • The presence of either pests or pesticides can lead to genotyping failures
  • For plant species whenever possible, collect young tissue, such as newly emerged leaves, rather than older tissue, as this tends to increase both the quantity and quality of DNA which can be extracted
  • For clients who have chosen to use previously extracted DNA:
    • Customers will receive much higher quality results from DNA extracted using either magnetic bead or spin column isolations
      • Kits are available from many reputable companies, including Qiagen, Thermo Fisher Scientific, and Promega. Freedom Markers has proven success with Qiagen MagAttract/Biosprint magnetic bead isolation and Qiagen DNeasy spin column isolations kits.
    • DNA should be quantified by florescence (e.g. Qubit or PicoGreen assays) whenever possible. However, Freedom Markers will accept absorbance (e.g. nanodrop) QC values. Please see below for required QC values
      • Florescence QC: 0.5 µg of DNA per sample
      • Absorbance QC: 7.5 µg of DNA per sample with a 260/280 ratio of 1.7-2.0 and 260/230 ratio of 2.0-2.2
Shipping Requirements:
  • Preferred – Shipping Tissue
    • Place lyophilized plant tissue in the provided 96 well plate(s) & seal with strip caps (view example)
    • Collect second rep of plates whenever possible
    • Wrap plate(s) in Saran Wrap and tape the wrap to ensure strip caps will not come off during shipping
    • Include a ziplock bag with additional tissue from the same species at the sample developmental stage as the actual samples
    • Ship lyophilized samples at room temperature to the address listed on the quote
    • If it is not possible to ship lyophilized samples, please send fresh samples using overnight shipping with *refrigerated* cooling block
      • NO ice, dry ice or frozen blocks should be used. Frozen samples received at the facility with be discarded
  • Alternative - Shipping DNA
    • In order to avoid cross-contamination of wells in plates, use strip caps/plugs and not sealing film
    • Wrap plate(s) in Saran Wrap and tape the wrap to ensure strip caps will not come off during shipping
    • Ship DNA samples overnight, frozen, and on dry ice to the shipping address listed on the quote
Some Other Important Details to Note:
  • Sample nomenclature
    • The Freedom Markers analysis pipeline requires samples to be uniquely named. Any samples with identical names will be assumed to be duplicated and merged into a single data point for genotype calling
    • Any non-alpha-numeric characters present in submitted sample names will be replaced with underscores
    • All sample names will be converted to UPPERCASE
  • Individual samples that fail QC cannot be replaced. If a significant number of samples fail QC in a given plate, customers will be given the option of recollecting and shipping all samples within the failed plate or proceeding with genotyping as is
  • If a substantial number of samples fail QC and it is not possible to collect replacements, the project will be concluded and the client will be invoiced for only DNA extraction & QC work performed to that point
  • Freedom Markers reserves the right to retain anonymized data for internal QC and process improvement
Project Deliverables:

At the completion of your project, a PDF project report will be made available to you, along with files of all figures and tables from the report.

As part of your project you will receive a SNP set from your samples. These SNPs were identified using a custom bioinformatic pipeline that we have spent years optimizing for tGBS® genotyping-by-sequencing (GBS) sequencing data. For Advanced and Comprehensive genotyping options only, we also provide the tGBS® genotyping sequence data for your individual samples. If you decide to try SNP calling from these data yourself we cannot provide advice on trimming, alignment, cut-offs etc. or make recommendations about bioinformatics software and the like because our recommendations would simply converge on the results that what we have already provided to you.

Other files will be made available to you depending on what Service Tier you selected

  • Individual FASTQ Files (Advanced and Comprehensive)
  • BAM (Alignment) Files (Advanced and Comprehensive)
  • Imputation (Comprehensive)
Reactivate Data Downloads:

After download links expire, in some but not all cases the dataset can be retrieved and reposted for download by the customer. When it is possible to recover data after the expiration on the regular data transfer window, a $25 recovery and reactivation fee will need to be paid. We strongly urge customers to download and backup datasets as soon as possible once finalized results become available.