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.

Order

Order in tube format

Order in plate format

General FAQ

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. (OPTIONAL) .fastq file - raw data on reads. Email support to request fastq files.
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.

Linear / Amplicon Sequencing FAQ

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.

Bacterial Genome Sequencing

Coming soon!