Sample Preparation

Concentration

Incorrect concentration is the leading cause of failures. Kindly submit your standard, substantial, or oversized plasmid samples adhering to the specified concentration and minimum volume provided in the table above. Quantify the samples using a Qubit or an equivalent fluorometric method, such as a plate reader. We do not recommend using Nanodrop for quantification because it is not reliable enough for the sequencing equipment.

Meticulous and precise quantification, followed by proper normalization, is paramount for successful sequencing. Over or under concentrating samples will most likely have an adverse impact on the results.

Quality

For optimal outcomes, strive for the presence of pristine, circular double-stranded plasmids. Plasmids that exhibit degradation or fragmentation pose a higher risk of sequencing failure, as they may fail to yield a consensus due to a lack of complete sequencing reads.

To verify size, conduct assessments on full-length plasmids (not digested or amplified) through gel electrophoresis. For linearized plasmids, employ a linear ladder, while intact circular plasmids benefit from a supercoiled ladder. It's essential to note that Sanger sequencing and PCR amplification are insufficient for size verification since these methods rely on primers to detect specific small regions.

The workflows for significant plasmids (25 - 125 kb) and extensive plasmids (125 - 300 kb) exhibit greater resilience to degradation, given the inherent difficulty in extracting plasmids of these larger sizes without some degree of degradation. Nonetheless, to ensure optimal results, the objective remains intact circular DNA even within these more forgiving workflows.

The linear/amplicon workflow exhibits greater resilience to degradation compared to the plasmid workflow, owing to its incorporation of minimal fragmentation during the library preparation process. Nevertheless, to achieve optimal outcomes, the pursuit of intact linear DNA molecules remains a paramount consideration.

Purity

The cleaner the product, the better the results. Leftover material in the sample can act as an inhibitor during sequencing. We suggest opting for samples exhibiting a 260/280 ratio exceeding 1.8 and a 260/230 ratio falling within the range of 2.0-2.2. Purity assessments can be conducted using Nanodrop or spectrophotometric methods, although these methods cannot be used to accurately measure the concentration.

For optimal outcomes, it is imperative that samples do not include any of the following components:

• Denaturants, such as guanidinium salts, phenol, etc., or detergents like SDS, Triton-X100, etc.
• Leftover contaminants from the organism.
• RNA (we recommend RNase treatment during extraction for its removal)
• Any insoluble material that would cause colors or cloudiness.

Furthermore, samples should contain copies of a single clonal molecule. You can send mixtures but it may yield inconsistent results. Submit mixtures at your own risk.

Questions on Whole Plasmid Sequencing

How fast is your turnaround time?

Currently >90% of results are delivered the same day the samples are received. The exact time varies but typically it ranges between 5PM - 11PM EST.

Is there a minimum number of samples I must submit?

Any number of samples is welcome. There are no minimums or limitations.

How can I ship my samples to you?

Dropboxes are available in many locations. Also, Eurofins is the only sequencing company that offers free digital shipping labels on >95% of orders. Take a look at our sample submission page for details.

How accurate is it?

The equipment vendor reports accuracy of 99.3%. The research community has reported accuracy between 93-98%.

How does whole plasmid sequencing compare to Sanger sequencing?

There are pros and cons of every sequencing method. Sanger sequencing and Oxford Nanopore sequencing (ONT) are both methods for determining the sequence of nucleotides in a piece of DNA. Typically Sanger is consider the most accurate method for short-read sequencing and NGS is better for long-read sequencing.

Overall, the choice between Sanger sequencing and ONT will depend on the specific needs of the application. Both methods have their strengths and limitations, and the appropriate method will depend on factors such as the length and quality of the DNA sample, the desired level of accuracy, and the cost and availability of the necessary equipment.

	Pros	Cons
Short read sequencing (Sanger)	Widely used and well-established method High accuracy and precision Can be automated for high-throughput sequencing Chromatogram makes it easy to visually interpret results	Limited read length (typically up to 1000 base pairs) Requires relatively large amounts of high-quality DNA Requires reassembly for covering longer regions. Difficult to sequence repetitive regions without gaps and resolve large variations.
Long read sequencing (Whole Plasmid)	Long read lengths (up to several hundred thousand base pairs) Can sequence a wide range of sample types, including low-quality and degraded DNA No primer design required All data from one sample	Lower accuracy compared to Sanger sequencing, particularly for shorter reads More expensive per base pair compared to Sanger sequencing ·Cannot resolve single bases.

Can I use my free barcodes on whole plasmid sequencing orders?

Absolutely. Using our free barcode labels is not required, but always an option.

Can I use my EVOcard to pay for my order?

Absolutely, yes.

Can you sequence my mixture of plasmids?

You can send it and we can sequence it, but we cannot predict or promise the analysis outcome. The customer would take on the risk of these orders.

Are there notable differences between Whole Plasmid Sequencing and Amplicon Sequencing in terms of the ordering process, submission guidelines, and result files?

The process is almost identical between Whole Plasmid Sequencing and Amplicon Sequencing.

1. Ordering is the same, using the same order pages.
2. Results are the same, with Amplicon sequencing offering all the same file types.
3. The sample submission guidelines is the same for both.
4. The turnaround time is generally the same.

How do I place an order?

It is easy. Go to our online ordering page.

Place order

Can I order through my institutions marketplace or B2B punchout?

We will be rolling out the new order page across portal sites, marketplaces, and punchouts very soon. At the moment, please submit orders on the main www.eurofinsgenomics.com website.

What is the coverage?

It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length plasmids and how many, if any, degraded.

What data files is delivered?

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.

What if my samples fail?

The probability of failure is low when using WPS, although it can still happen. If you wish to resequence a failed sample, contact Genomics Support. Unfortunately, we must charge for failed samples since it requires more time and energy than a normal run on the machine. If the sample fails a second time, all data points to a problem with the sample and we cannot resequence. In that scenario, we advise the customer to either send a new sample or troubleshoot the sample from their end.

Sample Preparation

Sample preparation for Linear / Amplicon samples is the same as whole plasmid samples. Refer to the WPS section for details.

Questions on Amplicon Sequencing

This section only contains answers that are different for Amplicon sequencing. Please check the General FAQ section for further information.

Can you sequence my mixture of plasmids?

You can send it and we can sequence it, but we cannot predict or promise the analysis outcome. The customer would take on the risk of these orders.

Are there notable differences between Whole Plasmid Sequencing and Amplicon Sequencing in terms of the ordering process, submission guidelines, turnaround time, and result files?

The process is almost identical between Whole Plasmid Sequencing and Amplicon Sequencing.

1. Ordering is the same, using the same order pages.
2. Results are the same, with Amplicon sequencing offering all the same file types.
3. The sample submission guidelines is the same for both.
4. The turnaround time is generally the same.

What is the sequencing coverage for linear/amplicon?

It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length plasmids and how many, if any, degraded. Generally speaking, the coverage for linear/amplicon samples is similar to whole plasmid sequencing.

Why are there errors or low confidence positions in a homopolymer region?

Prevalent error patterns in Oxford Nanopore sequencing often manifest as deletions within homopolymer stretches and inaccuracies occurring specifically at the central position of the Dcm methylation site, CCTGG or CCAGG. Anticipated advancements in sequencing chemistry and basecalling software updates are poised to ameliorate these limitations in the foreseeable future.

Why are there terminal nucleotides missing from my sequence?

The assembler occasionally encounters challenges in reconstructing the terminal ends of linear DNA, potentially leading to the omission of approximately ~25 nucleotides from the 3' and/or 5' ends of your insert, contingent upon your sample's sequence. Should you observe this occurrence, you have the option to download the raw reads from your Dashboard and employ your preferred methodology to reconstruct the ends.

What if my samples fail?

The probability of failure is low when using WPS, although it can still happen. If you wish to resequence a failed sample, contact Genomics Support. Unfortunately, we must charge for failed samples since it requires more time and energy than a normal run on the machine. If the sample fails a second time, all data points to a problem with the sample and we cannot resequence. In that scenario, we advise the customer to either send a new sample or troubleshoot the sample from their end.

Sample Preparation

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.

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.

Questions on Bacterial Genome Sequencing

This section only contains answers that are different from the information above. Please check the General FAQ section for further information.

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.

Please visit the shipping samples page for all information on physically submitting samples.