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.

What is the difference between Bacterial Genome Sequencing and other bacterial sequencing options on the order page?

Customers can submit bacterial samples using either option. The only difference is that Bacterial Genome Sequencing uses a different bioinformatics pipeline and kit to process more data, while the other bacterial options (Bacterial colonies, Glycerol Stock/Bacterial Cultures, and Pellets) use the standard ONT pipeline. You can still submit colonies, cultures, and pellets for the Bacterial Genome Sequencing service, simply pick Bacterial Genome Sequencing and submit. If you choose one of the other options, you are telling the lab what type of sample to expect and choosing the standard pipeline.
 

Online option	Pipeline
Bacterial Genome Sequencing	> Special pipeline
Bacterial Colonies	> Standard pipeline
Glycorol Stock / Bacterial Cultures	> Standard pipeline
Bacterial Pellet	> Standard pipeline

What is your turnaround time for bacterial genome sequencing?

Typically 2 days. If extraction is required, the turnaround time is 1 week. In rare instances, it may take longer depending on the complexity of the project and the volume of samples being processed.

Are there notable differences in the order process between bacterial genome sequencing and the other ONT services?

The process is almost identical. Go to the normal order page and you will see an option to select bacterial genome in second step of the order process.

What is the accuracy of this service?

Oxford Nanopore touts accuracy of 99% raw read accuracy. The scientific community has reported results ranging from 93-98%. Final assembly is contingent upon both coverage and data quality. A higher coverage, denoting an increased number of reads used to construct a consensus, typically augments the accuracy of the obtained results.

What defines successful sequencing for bacterial genomes?

The minimum targets for successful sequencing can be found on the bacterial genome webpage. In short, the target is 30x coverage which equates to 210 Mb for medium size samples and 360 Mb for the larger size.

What are the data deliverables for bacterial genome sequencing?

All the same data files are provided as found with whole plasmid and amplicon sequencing, plus an additional report.

1. .fasta file (for consensus data): we will provide a clean, complete consensus sequence for each plasmid.
   
2. .gbk GenBank file (for consensus data): a pLannotate map in the GenBank file format.
3. .fastq file - raw data on reads.
4. Histogram file: the hisogram file provides a visual representation of the plasmid and raw read data for deeper insight into your samples (image).
5. .html pLannotate map (for consensus data): a plasmid map for each sample.
   
6. .csv confidence file with quality statistics.
7. Comprehensive report: an example can be downloaded and viewed from the sidebar.
   

What is your repeat policy for bacterial genome sequencing?

If none of the targets set for successful sequencing are met, then our technicians will evaluate the results to determine the feasibility of achieving a more successful outcome a second time. If there is potential for success, typically a repeat is run at no additional cost. Customers may submit a repeat request for evaluation as well. However, should the desired outcomes not be attained during the second attempt, or if our technicians determine that the potential for success in a second run is low, then further repeats will not be initiated. Should you opt to sequence the sample anew, it is imperative to prepare fresh samples that adhere to all quality control (QC) requirements before submitting a new sequencing request.

Can you sequence my mixture of different bacterial species?

This service is designed for the analysis of clonal populations, specifically a single species of bacteria. While it is permissible to submit mixtures of different bacterial species for sequencing, predicting the assembly outcome is challenging, and therefore, it is undertaken at your own risk. The total raw data acquired for your sample will be proportionally allocated among the different species present. Consequently, this division diminishes the individual genome coverage of each species, potentially impeding the assembly of specific species within the sample. Re-sequencing mixtures does not alter the relative proportions of the species. However, if higher total coverage is needed, multiple aliquots can be submitted. The ultimate determination of which species yield an assembly depends on the overall sample quality, coverage, and the relative abundance or degradation of each species.

Is it possible to sequence the bacteria’s native plasmid at the same time?

Should your genomic DNA (gDNA) extraction encompass native plasmid DNA, it is likely that corresponding sequencing reads for these plasmids will be obtained. The standard sequencing procedure typically excludes input DNA fragments smaller than 3kb. However, we do not selectively filter out diminutive plasmid-sized reads during the assembly process. Consequently, it is probable that these smaller reads will contribute to the creation of distinct plasmid assemblies alongside the overarching gDNA assembly. The ultimate outcome, determining which DNA types within your sample contribute to the assembly, hinges on factors such as overall sample quality, coverage, and the relative abundance or degradation of each DNA type.

Is it possible to sequence a genome that is linear, multi-chromosomal, and/or over 12 Mb?

Certainly, any species is technically amenable to sequencing and assembly using this method. However, it is essential to acknowledge that submitting samples for applications beyond bacteria entails inherent risks. This is due to the fact that we have not fine-tuned the optimal data requirements for various specimen types, and our assembly/annotation pipeline is primarily tailored for bacteria.

For non-bacterial applications, there may be a necessity to submit multiple aliquots of each sample to ensure adequate genome coverage for larger and more intricate eukaryotic genomes. It is important to highlight that data from all aliquots must be amalgamated before initiating your assembly pipeline.

In the event of planning to submit a substantial number of samples for such off-label applications, we strongly recommend reaching out to us prior to submission. This proactive communication will facilitate discussions on viable options and ensure a more informed approach to meet your specific requirements.