Whole Plasmid Sequencing

Description of Service

The Whole Plasmid Sequencing service is intended for the full-length sequencing and annotation of clonal, circular, double-stranded plasmid DNA up to 300 kb in length. In the vast majority of cases, we deliver plasmid sequencing results within one business day of receipt of your samples.

This service is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies , and includes the following components:

  • Constructing 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.
  • Sequencing the library with a primer-free protocol using the most accurate R10.4.1 flow cells (raw data is delivered in .fastq.gz format).
  • Generating a high-accuracy circular consensus sequence from the raw reads.
  • For standard size plasmids, we will also return a set of feature annotations.

This service is intended for a clonal population of molecules. If there are multiple molecular species present, the pipeline will only return one consensus sequence for the single molecular species that produces the largest amount of total sequencing data. If you would like to sequence a known mixture (e.g. barcode or variant libraries), please consider submitting instead to our Custom Sequencing Service. Please read more the topic of sequencing mixtures in our Results Interpretation Guide.

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 amounts of data produced by 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.
good histogram

Fig2. A histogram with multiple peaks indicates unexpected products, deletions, recombinations, or concatemers

Data & File Types Delivered

When your results are ready, you will receive an email notification. Once you sign in to your account, you can download these results from your Dashboard. More detailed information about data & file types can be found in the Results Interpretation Guides.

  • Consensus sequence (.fasta file): Polished consensus sequence of the plasmid.
  • Consensus sequence (.gbk file): Polished consensus sequence of the plasmid, with a plasmid map and feature annotations.
  • Plasmid map (.html file): An interactive version of the plasmid map.
  • Read length histogram (.png file): Displays the read length distribution of the raw reads produced by your sample.
  • Virtual gel (.png file): Displays the raw read lengths from all samples in the order in a virtual gel format.
  • Chromatogram (.ab1 file): Displays the relative abundance of each nucleotide (A, T, G, C) for all raw reads that align to the consensus at each position of the sequence.
  • Coverage plot (.png file): Displays the relative sequencing coverage at each position of the consensus sequence.
  • Per-base data (.txt and .tsv files): Includes 3 sub-files for each sample:
    • SAMPLE.tsv: Indicates how well the raw reads agree with the consensus sequence at each position.
    • SAMPLE_multimer_analysis.txt: Indicates the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.).
    • SAMPLE_summary.tsv: Indicates the length, average coverage, relative composition (by moles and mass), total reads, total bases, and %. E. coli genomic DNA contamination for the consensus sequence.
  • Raw read sequences (.fastq.gz file): Provides the sequences of individual raw reads that align to the consensus. Please note that these reads are NOT delivered in the default download, but can be downloaded separately by clicking the Download Raw FASTQ button at the top of the Order Information page. Also note that any raw reads that do not align to the consensus (e.g. host genomic DNA, lower abundance molecular species) are excluded.
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.

Preparing Your Purified Plasmid Samples

This service requires clonal, circular, double-stranded plasmid DNA at the minimum volume and within the concentration range specified during order submission in your Dashboard.

Single-stranded DNA is not currently a supported application for this service. Some customers do send ssDNA to this service, but the results are highly variable and we cannot guarantee success; if you opt to submit ssDNA, please be aware this would be at your own risk.

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 DMSO 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 at least the minimum volume of your plasmid samples within the concentration range specified during order submission in your Dashboard. For best results, we strongly recommend performing DNA quantification assay with Qubit or equivalent fluorometric method (such as a plate reader).

If you opt to use a spectrophotometric method like Nanodrop, please be aware that these methods are much less accurate for measuring DNA concentration. 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.

CAUTION: Sanger sequencing and PCR amplification are NOT adequate for full-length size verification because these methods use primers to detect only a specific small region, so they cannot provide information about the integrity of your entire plasmid molecule.

NOTE: 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 as much intact circular DNA as possible.

We recommend samples with 260/280 above 1.8 and 260/230 between 2.0-2.2. Purity may be assayed with Nanodrop or other spectrophotometric methods. If using a Qiagen miniprep kit, we recommend performing the optional PB buffer wash step to increase purity.

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 is not a supported application and is at your own risk!

CAUTION: Sanger sequencing and PCR amplification are not adequate to verify clonal purity because they use primers that bind only to a specific small region, meaning a positive signal may be obtained even if a very small fraction of the total molecules actually contain the target sequence. This may artificially create the appearance of a pure clonal 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 does not use any primers and will produce raw reads data for all molecular species present in the sample, and is therefore a much more accurate depiction of the true contents of your sample.

Interpreting Your Results

Please refer to our Results Interpretation Guides for details.