Best Practices for Shipping Samples and Libraries for Next Generation Sequencing

⬅️ NGS Handbook

One of the most frequently asked questions by researchers ordering services on Genohub is what is the best storage and shipping solution to ship extracted nucleic acid samples or libraries. What you tend to hear from each lab manager can vary, so we decided to put together a unified standard. With over 100 service providers offering library preparation and sequencing services on Genohub shipping recommendations were diverse, but going through each one of these we’ve put together a set of best practices. If you’re ordering a sequencing service on Genohub or even from a provider outside of Genohub’s network, you can follow these guidelines and rest assured that your samples will have been prepared and shipped properly.

Shipping RNA Samples

Dry ice is by far the easiest and most recommended way to ship total RNA samples. If you can’t ship with dry ice an ethanol precipitate is also fine. If you have an absolutely pure total RNA sample you can probably ship at room temperature or leave the sample on your bench for 1 week. However the reality is that many preparations and isolations do not completely remove nucleases. If your sample is contaminated with nucleases, you’ll need to slow down the rate it chops your RNA by reducing temperature. Finally, remember that RNA can be fragmented in water with divalent cations and heat, so heating RNA in water is not a good idea either. See our recommendations for shipping RNA in dry ice (recommended) or an ethanol precipitate if you can’t ship with dry ice.

Dry Ice

For most RNA applications, you’ll need to ship total RNA. If you’re interested in small RNA species, don’t forget to use a method that preserves small RNAs. Clean the surface of all tubes and use nuclease-free tips. Use nuclease-free water for buffer preparation and reconstitution of your total RNA sample. See the Packing section below and follow instructions for shipping with dry ice.

Ethanol Precipitation

Re-suspend your total RNA in a 100 uL precipitate solution.

Add:

  1. 1/10th volume of 3M NaOAc, pH 5.2
  2. 3 volumes of 100% ethanol

Mix well by vortex. Follow packing instructions below, but instead of dry ice use wet ice packs to maintain a -20 to 4°C shipping temperature.

Shipping DNA Samples

Shipping DNA that has been dissolved or re-constituted in TE (10 mM Tris, pH 8, 0.1 mM EDTA) or 10 mM Tris pH 8.0 with 4°C wet ice packs (blue ice) is the recommended way to ship DNA samples to a service provider who will make your libraries. Some service providers worry about the EDTA in TE inhibiting enzymatic reactions during library prep. If you’re only using 0.1 mM EDTA, by the time your concentrated DNA is diluted, and then diluted again during the first step of library preparation, the concentration of EDTA is so low, there is no chance it’s going to have any effect on the enzymes in library preparation. If after this, you’re still worried, just reconstitute your DNA in 10 mM Tris pH 8.0 rather than TE.

Yes, you can probably get away with shipping a pure DNA sample in water at ambient temperature. You can also lyophilize your DNA or dry it down on paper, but make the life of your service provider easier and just ship on a wet ice pack. You can ship extracted DNA on dry ice, but there is no good reason for this.

Shipping Fresh Tissue

Fresh tissue should be snap frozen and shipped on dry ice. If you’d like a provider to extract RNA or DNA from this tissue or if you can’t ship on dry ice, a preservation solution is recommended, e.g. RNAlater™ or DNA/RNA Shield™. These are aqueous solutions composed of ammonium sulfate that co-precipitate RNA and cellular proteins, 1) rendering RNA physically inaccessible to nucleases, and 2) inhibiting nucleases. Ammonium sulfate precipitation is typically a method used for purifying proteins by altering their solubility. Protein solubility depends on the ionic strength of the solution. At low ionic strength, solubility of protein increases with increasing salt, at high ionic strength, protein is completely precipitated out of solution, also known as ‘salting-out’.

There are a few online protocols for making your own ammonium sulfate based precipitation solution. Here is an example.

Commercial or homemade RNA preservation will preserve RNA for up to 1 week at room temperature or 4 weeks at 4°C, allowing processing and shipping of samples without liquid nitrogen or dry ice.

Shipping Plasma Samples

For the shipment of plasma samples, please contact your shipping courier beforehand. Plasma samples are often considered "Clinical Samples" and you will need to label them as "Exempt Human Samples" to avoid them being considered biohazardous materials. “Exempt human or animal specimen” means a human or animal sample (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) transported for routine testing not related to the diagnosis of an infectious disease. Typically, exempt human specimens are specimens for which there is a low probability that the sample is infectious, such as specimens for drug or alcohol testing; cholesterol testing; blood glucose level testing; prostate-specific antigens (PSA) testing; testing to monitor heart, kidney, or liver function; pregnancy testing; and testing for diagnosis of noninfectious diseases such as cancer biopsies.

Constructed Library

A constructed library is a very stable, double stranded PCR product. Most providers will want between 2 – 10 nM of that library for sequencing. If you’re shipping a library you’ve constructed yourself, reconstitute your library in ~10-50 µl of 10 mM Tris pH 8.0 buffer and use microcentrifuge tubes that are 'low-bind'. At very low concentrations, libraries tend to stick to tube walls.

Our recommendations for shipping constructed libraries are the same ones as shipping DNA samples.

Packing and Shipping

Although most providers will have specific recommendations and preferences specified in the Terms & Procedures tab of your Genohub project, below are some general guidelines to follow when shipping.

Place your DNA or RNA sample in a 1.5 or 2 mL screw cap microcentrifuge tube and seal with Parafilm. Pack the tube in a freezer box, 50 mL conical vial or some other method to protect it from breaking. Place into a thermo-stable shipping box. We recommend the Styrofoam be at least 1.5 inches thick. Fill the box with dry ice or wet ice. See RNA or DNA sections in this guide to determine whether dry ice or wet ice is recommended. Here are our rough recommendations for the quantity of ice to pack:

  • Shipments within your continent: 4 kg
  • Shipments between Europe and North America: 6 kg
  • Shipments between Asia and North America 9 kg
  • Shipments between South and North America 9 kg

In a thermostable box where the Styrofoam is at least 1.5 inches thick, 3-4 kg of dry ice should last for at least 48 hours.

If you want to place the Styrofoam container into a standard shipping box, please use a new outer box, if possible. If you must re-use a box, then either remove or black out any pre-existing labels.

Use the fastest available courier, e.g. FedEx, UPS, DHL or USPS with a tracking number. The carrier may ask that you fill out a commercial invoice. Label your contents as ‘non-hazardous research sample’. Label the box as ‘temperature sensitive, keep frozen’. Send the service provider a tracking number so they know when to expect your package.

For international shipping, attempt to ship on Monday or Tuesday to avoid delays associated with delivering on a weekend. Determine whether the country you are shipping to has a holiday. Shipping during a holiday can needlessly delay the time it takes for a service provider to unpack and properly store your samples.

Using these recommendations will completely ensure you’re properly shipping raw, extracted nucleic acid, tissue or libraries. If you’re looking to find and compare sequencing and library preparation quotes between service providers, use Genohub’s NGS search interface. Once you’ve narrowed down the quotation you’d like to move forward with, your project is confirmed and detailed shipping instructions and conditions are displayed.

Shipping to the United States

Shipments from around the world to the United States frequently arrive without any issue or hold up at customs. A small percentage of shipments do get held. To reduce this chance, consult your local international courier to see what they require before you ship. To minimize the chance of any issues, include the following letter in your shipping documents both inside and outside the shipping container. Having the letter outside the container makes it more easily visible to U.S. Customs and/or USDA inspectors. Edit the letter based on your specific samples:

Contents of this package are non-Infectious, non-Hazardous, not an etiologic agent, and not for human consumption. Shipment consists of sterile DNA for scientific analysis only. The material contains small pieces of genomic DNA suspended in sterile water. The material was not generated by microbial fermentation. The product is purified and does not contain animal or cell derived materials or additives. The material is non-toxic, non-hazardous and nonpathogenic. It contains no known pathogens or viruses and is not hazardous to human health. It is not a biohazard, and in case of damage and release, it will not affect other organisms. It is safe for air transport and all substances comply with applicable regulations. The end use is for research purposes only, namely the generation of sequencing data by:

[ENTER NAME, ADDRESS and PHONE NUMBER of SERVICE PROVIDER]

and will be used for laboratory research purposes only. Material will be maintained within appropriate biological containment facilities, preventing exposure of material to plants, animals or the public. This package is temperature sensitive and needs to be handled in a timely manner.

[ENTER your NAME AND CONTACT INFORMATION HERE]

You may also need to complete an invoice (commercial invoice, customs invoice or pro-forma invoice) for customs and include it with the shipment. Your local international courier should have more information about this requirement.

Please see below for examples of what to include in the invoice, if requested. Edit based on your samples:

  • “RNA or DNA Samples for Research Use Only”
  • “Non-Dangerous, Non-Infectious”
  • “No Commercial Value, Value for Customs Only”
  • Declare the value of the goods for customs (e.g. $1 USD). Note: The declared value should not be more than $10 USD to make sure it’s not flagged as a purchase or merchandise.

If the FDA puts a hold on the package, you may need to provide the following:

  • Country of Origin
  • Scientific Name
  • Manufacturer (e.g. your institution)
  • Intended Use of Goods (e.g. routine lab test for research purposes)

If U.S. Customs puts a hold on your package, you may need to check if an import permit is needed. Please see the following websites for USDA import information.

You may need to generate a letter similar to the one below. Please edit based on your samples and the correct USDA Guideline.

As per the USDA Guideline 1120 (https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-and-animal-product-importinformation/import-live-animals/no-import-permit-req) this sample set:

  1. Is not derived from livestock or avian species;
  2. Is for in vitro use only;
  3. Has not been exposed to livestock or avian disease agents exotic to the United States; and
  4. Does not produce antigens or contain genes of livestock or avian disease agents
  5. Does not produce monoclonal antibodies directed against livestock or avian disease agents.

Therefore, this shipment may be imported without a USDA permit.

Other Resources: