Direct Colony Sequencing ONT

I. Bacterial Genomic DNA (gDNA) Samples

To facilitate the seamless execution of this service, it is imperative that customers provide 1 µg of high-quality, high-purity, and high-molecular-weight (HMW) double-stranded genomic DNA (gDNA). The optimal specifications include having more than 50% of the DNA exceeding 15 kb in length, with a recommended purity ratio of 260/280 surpassing 1.8 and the 260/230 ratio falling within the range of 2.0-2.2.

Notably, our affordable pricing and rapid turnaround times do not encompass quality control (QC) services for incoming samples. Hence, it becomes the submitter’s obligation to ensure that the prepared samples adhere to these stipulated requirements before shipping. This proactive verification step is essential to guarantee the successful processing of your bacterial gDNA samples.

Sample prep steps

1. Prepare sample from a bacterial clonal culture
   • There are various methodologies available online in the public domain for how best to prepare bacterial cultures. We encourage researchers to seek out the protocols that fit their needs.
2. Extract and purify sample from clonal culture
   • Again there are many different extraction methods available on the market. Eurofins Genomics is indifferent to the type of extraction method as long as it produces high-quality, high-purity, high-molecular-weight (HMW), double-stranded genomic DNA (gDNA) devoid of nicks, gaps, breaks, and contaminants is deemed suitable for this sequencing service. Here are a few recommendations for extraction kits from trusted brands:
   • Zymo
     • Zymo® Quick-DNA Miniprep Plus Kit
     • Zymo® Quick-DNA Fungal/Bacteria Miniprep Kit.
   • Wizard - Wizard® Genomic DNA Purification kit
   • Qiagen
     • Qiagen® MagAttract HMW kit
     • Qiagen® DNeasy kit
   • Additional Tips
     • Refrain from vortexing.
     • Use wide-bore tips for pipetting.
     • Elute in elution buffer instead of water.
     • Avoid exposure to high temperatures (>37°C) for more than 1 hour, extreme pH levels (<6 or >9), intercalating fluorescent dyes, or UV radiation.
     • Steer clear of freeze-thaw cycles; store gDNA at 4°C for 1-2 months.
     • If utilizing a speed-vac, avoid heat and be careful not to over-dry.

3. QC the sample before shipping it

This step involves 3 areas: quantity, quality, and purity.

Quantity: It is imperative to provide 1 µg of genomic DNA (gDNA) at a concentration of 50 ng/µL in 20 µL of elution buffer. We recommend using a Qubit for quantification or another fluorometric method, such as a plate reader, and we discourage the use of Nanodrop.

For high molecular weight (HMW) gDNA, additional homogenization efforts, such as an extended incubation time, elevated incubation temperature, and thorough, gentle mixing, may be necessary for precise quantification. Adequate homogeneity is typically indicated when separate DNA quantifications from the top and bottom of the sample differ by less than 15%.

If <1 µg was obtained from the first extraction, we highly recommend performing additional extractions to meet the yield criteria. You can submit <1 µg but at your own risk. If submitting <1 µg, prepare the sample at the required concentration (50 ng/µL) but in a reduced volume based on your total yield. Also, an email to GenomicsSupport@eurofins.com prior to shipping and always appreciated so we know what to expect.

Quality: over 50% of the total DNA should be above 15 kb in size. If not, we highly recommend extracting and purifying again. There are multiple options for size characterization, including Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder.

Purity: the minimum purity for gDNA samples is 260/280 ratio above 1.8 and a 260/230 ratio between 2.0-2.2. Acceptable options for testing purity include Nanodrop or other spectrophotometric methods. If the sample does not meet the recommended criteria, please re-extract or cleanup using a Qiagen cleanup kit or AMPure XP beads.

Additional Tips

- No RNA. The best way to prevent RNA is to use an RNase treatment during extraction.
- No denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.).
- No residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.).
- No insoluble material or exhibit coloration or cloudiness.

II. Cell Pellets for the Bacterial DNA Extraction Option

Cell pellets from both BSL1 and BSL2 strains are allowed. Pellets should be reconstituted in Zymo 1X DNA/RNA Shield. Furthermore, we encourage customers to cultivate a freshly grown clonal culture of your bacteria in liquid broth. The best time to harvest cells is doing the growth stage or early stationary phase. Sending cells from older cultures in the death phase is discouraged.

Please note that Eurofins Genomics does not cultivate samples. Our lab will extract DNA from the material you submit. Without adequate cell collection from the culture, the extraction process is likely to fail. Lastly, this service is only available in the US due to customs regulations.

Sample Prep Steps

1. Prepare the cell pellets
   1. Centrifuge the cells into pellets to remove excess supernatant. A compact cell pellet should weight approximately 15 mg and not exceed 50 mg. The requisite quantity is equivalent to 8-12 OD600 or 4-6 x 10^9 cells (e.g., 8-12 mL culture at 1.0 OD600).
      1. In the case of wet cell pellets (e.g., Streptococcus sp.), where complete removal of supernatant without disturbing the pellet is not feasible, an approximate weight of 30-50 mg is recommended.
   2. Resuspend in 1 mL PBS, followed by another round of centrifugation to remove further supernatants.
   3. Conclude the process by resuspending the pellet in 0.5 mL of Zymo 1X DNA/RNA Shield inside a 2 mL tube.

2. Submit order
   1. Go to the order page and select the correct size. .
   2. Be sure to print the confirmation page and folder it inside the tube bag when submitting/shipping the samples. Ensure that each tube is clearly labeled with the order code, and sample number.

3. Ship sample
   1. There are multiple options for how to submit samples, including dropboxes, digital shipping labels (provided free during checkout for all orders >$30), or ship using your own shipping carrier.
   2. To safeguard against potential damage and leakage during transportation, it is essential to position the screw cap tubes within a sturdy container, such as a falcon tube or tube box, before dispatching. For orders exceeding 10 samples, specifically arrange the tubes within a tube box, loading the samples row by row in numerical order. This meticulous organization significantly streamlines the sample reception process, reducing handling time. Please dispatch the samples at room temperature for optimal conditions.

Sample Submission Guidelines

• Submitting in colony
  • Wrap agar plates in parafilm for submission.
  • Circle the colonies you wish to pick using a sharpie marker. If nothing is drawn, we will take this to mean 'pick at random'.
  • Please submit only E. coli containing plasmids; high-copy plasmids in standard cloning strains yield the best results.
• Submitting in plates
  • Clearly label plates with the order tracking number.
  • Circle the wells you wish to pick using a sharpie marker. If nothing is drawn, we will take this to mean 'pick at random'.
• If submitting multiple plates at once, make sure the sample names are clear and labeled, e.g., Plate1-Colony1.
• Ship at room temperature in padded container. .

The main difference is the sequencing service. Bacterial Genome Sequencing uses a different bioinformatics pipeline and kit designed for the comprehensive analysis of entire genomes, while Direct Colony Sequencing uses the standard ONT sequencing pipeline combined with rolling circle amplification (RCA).

Customers can submit bacterial samples using either option. Both services accept the same sample types.