Linear/Amplicon Sequencing


Sample type Category Size Concentration Minimum volume Price per sample Order now
Linear/Amplicon Standard 600 bp - 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
Dinocoins Pre-paid credits $15 Purchase

Description of Service

The linear/amplicon sequencing service is intended for the sequencing of clonal linear DNA from 600 bp to 125 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 minimal fragmentation of the linear 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).
  • Re-assemble the raw reads and align them against each other to generate a high-accuracy linear consensus sequence.

You will receive the following data for whole plasmid sequencing:

  • .fasta file (for consensus data): We return a polished consensus sequence of each linear/amplicon 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.

We also 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.

    sample .fastq file

    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 linear/amplicon 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 Linear/Amplicon DNA Samples

This service requires 300 ng (standard linear/amplicon) or 1000 ng (big linear/amplicon) of linear double-stranded DNA, normalized to the specific concentration listed in the table above.

Submit the final purified linear/amplicons 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.

Linear/amplicon 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 or big linear/amplicon 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!

This service is intended for linear double-stranded DNA molecules.

Size verification should be performed on full-length linear/amplicon DNA via gel electrophoresis.

The linear/amplicon workflow is more tolerant to degradation than the plasmid workflow because it does include some minimal fragmentation during the library prep process (and thus we do not return read length histograms for linear/amplicon samples because a clonal peak is not produced and it is typically not informative). However, for best results, you'll still want to aim for intact linear DNA molecules.

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 molecule. Sending mixtures of molecular species will give mixed results and is at your own risk!

Shipping Your Linear/Amplicon Samples

Please refer to our shipping instructions for details.

Linear/Amplicon Sequencing FAQ