plasmidsaurus

Whole Plasmid Sequencing

Sample type	Category	Size	Concentration	Minimum volume	Price per sample	Order now
Plasmid	Standard	2.5 - 25 kb	30 ng/uL	≥10 uL	$15 or 1 dinocoin	Order
	Big	25 - 125 kb	50 ng/uL	≥20 uL	$30 or 2 dinocoins	Order
	Huge	125 - 300 kb	50 ng/uL	≥40 uL	$60 or 4 dinocoins	Order
Dinocoins	Pre-paid credits				$15	Purchase

Description of Service

The whole plasmid sequencing service is intended for the full-length sequencing and annotation of clonal circular plasmid DNA between 2.5 kb and 300 kb in length. This service is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies (ONT), and includes the following components:

Construct an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including linearization of the circular input DNA in a sequence independent-manner.
Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data is ~99.6% accurate and is delivered in .fastq format).
Align the raw reads against each other to generate a high-accuracy circular consensus sequence and a set of gene annotations.

You will receive the following data for whole plasmid sequencing:

.fasta file (for consensus data): We return a polished consensus sequence of each plasmid in .fasta format.
[OPTIONAL] .fastq file (for raw reads): If you like, you can elect during the order process to receive the raw .fastq sequencing reads delivered to your email. You can also download the raw reads anytime from your Dashboard.
.svg file (for raw reads): We return raw read length histograms for each plasmid, which provide unique insight into the contents of your samples. See details on interpreting your histograms.
.html pLannotate map (for consensus data): We return a plasmid map for each sample, generated with the excellent pLannotate tool from the Barrick Lab.
.gbk GenBank file (for consensus data): We return the same pLannotate map in GenBank file format.

pLannotate image — McGuffie,M.J. and Barrick,J.E. (2021) pLannotate: engineered plasmid annotation. *Nucleic Acids Research* DOI: 10.1093/nar/gkab374

Finally, we return two files that show how confident we are in our basecall at each position of our consensus sequence:

.csv file (comparing raw reads to consensus data): We align the raw reads to the consensus results and return a stats.csv file that lists how well the two datasets agree at each position.
.fastq file (comparing raw reads to consensus data): We create this .fastq file from the stats.csv file to visualize our consensus confidence. It contains the consensus base call and a “consensus score” (an internal metric of our confidence for the consensus basecall) for each position. This .fastq file can be viewed in software such as SnapGene Viewer to quickly identify low-confidence positions.

Shorter bars correspond to lower confidence positions.

Our ability to deliver these target outputs is directly dependent on the quantity, quality, and purity of the plasmid DNA sent to us, so we do not guarantee results. If we are not able to generate a consensus sequence from your sample, our failure policy applies.

Whole Plasmid Sequencing vs. Sanger Sequencing

Unlike traditional Sanger sequencing, which relies on primers to detect only a specific small region, our full-length ONT service sequences each entire plasmid molecule with a single long read. All molecules within a received sample are sequenced, including any degraded plasmids or background genomic DNA; we do not use any primers which would target specific regions or types of molecules.
As a result:

We are able to reveal structural issues that are not detectable with Sanger.

The relative read counts for different molecular species will roughly correspond to the actual proportions of those species within the sample.

If you find that our consensus does not match your reference, it is likely that the plasmid construct you sent us is missing elements, contains mutations, etc. compared to your reference. This is a common outcome revealed by whole plasmid sequencing!

good histogram — **Fig1.** A histogram with **one dominant peak** typically indicates a clean prep with a single plasmid.

Preparing Your Plasmid DNA Samples

This service requires 300 ng (standard plasmid), 1000 ng (big plasmid), or 2000 ng (huge plasmid) of circular, double-stranded DNA, normalized to the specific concentration listed in the table above.

Please refer to published literature for plasmid extraction protocols. Submit the final purified plasmids in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing EDTA (e.g. TE or AE buffer) whenever possible.

Plasmid samples are sequenced WITHOUT primers, so please DO NOT ship any primers with your samples or mixed into your samples.

Our low sequencing prices and fast turnaround times do not include DNA extraction or quality control (QC) services, so please verify with full QC that your samples meet the following requirements prior to shipping.

Submit your standard, big, or huge plasmid samples normalized to the specific concentration and minimum volume listed in the table above. Quantification assay must be performed with Qubit or equivalent fluorometric method (such as a plate reader).
IMPORTANT: Do not use Nanodrop for DNA quantification – spectrophotometric methods are NOT reliable for quantification!

Sending samples at too high OR too low concentration may adversely affect the library prep and/or sequencing reactions, possibly resulting in sequencing failure. Sending samples at the incorrect concentration (usually due to using Nanodrop for quantification) is by far the most common cause of sequencing failure. Accurate quantification and normalization are key!

For best results, aim for intact circular double-stranded plasmids. Plasmids that are degraded or fragmented are much more likely to result in sequencing failure by yielding no consensus due to lack of full-length sequencing reads.

Size verification should be performed on full-length plasmids (NOT digested or amplified plasmids) via gel electrophoresis; use a linear ladder for linearized plasmids, and a supercoiled ladder for intact circular plasmids.

Sanger sequencing and PCR amplification are NOT adequate for size verification because these methods use primers to detect only a specific small region.

The big plasmid (25 - 125 kb) and huge plasmid (125 - 300 kb) workflows are more tolerant to degradation because it's more difficult to extract plasmids of these larger sizes without some amount of degradation. However, for best results, you'll still want to aim for intact circular DNA.

We recommend samples with 260/280 above 1.8 and 260/230 between 2.0-2.2. Purity assay may be performed with Nanodrop or other spectrophotometric methods.
IMPORTANT: Do not refer to the DNA concentration reported by Nanodrop as a substitute for the above fluorometric quantification!

For best results, samples should NOT contain any of the following:

RNA (RNase treatment is recommended during extraction)
Denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
Residual contaminants from the organism (heme, humic acid, polyphenols, etc.)
Insoluble material, colors, or cloudiness

Samples should also be "pure" in the sense that they should only contain copies of a single clonal plasmid molecule. Sending mixtures of molecular species will give mixed results and is at your own risk!

Sanger sequencing and PCR amplification are NOT adequate for purity verification because they use primers that bind only to a specific small region. As a result, a strong Sanger sequencing signal may be obtained even if a very small fraction of the total molecules actually contain the target sequence. This may artifactually create the appearance of a pure sample that contains only one molecular species, when in fact other molecular species that lack the primer binding sequences may also be present but not detected. Our whole plasmid sequencing service will produce data for all molecular species present in the sample and is therefore a much more accurate depiction of the true contents of your sample.

Sometimes single-stranded circular DNA can also have successful results with this plasmid sequencing service. It's not an officially supported application though, so try it at your own risk.

Shipping Your Plasmid Samples