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
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
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
.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.
Finally, we return two files that show how confident we are in our basecall at each position of our
.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
.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
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
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
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!
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
Please refer to published literature for plasmid extraction protocols. Submit the final purified plasmids in
buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing EDTA (e.g. TE or AE
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
volume listed in the table above. Quantification assay must be performed with Qubit
fluorometric method (such as a plate reader). IMPORTANT: Do not use Nanodrop for DNA quantification – spectrophotometric methods are NOT reliable
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
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)
gel electrophoresis; use a linear ladder for linearized plasmids, and a supercoiled ladder for intact
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
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
or other spectrophotometric methods. IMPORTANT: Do not refer to the DNA concentration reported by Nanodrop as a substitute for the above
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.)
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.