Linear/PCR Sequencing

Description of Service

The Linear/PCR Sequencing service is intended for the full-length sequencing and annotation of clonal, linear, double-stranded DNA between 500 bp - 125 kb in length. In the vast majority of cases, we deliver linear/PCR 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 minimal fragmentation of the linear 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).
  • Using the re-assembled raw reads to generate a high-accuracy linear consensus sequence from the raw reads.
  • For standard linear/PCR samples, 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 Premium PCR Sequencing Service or Custom Sequencing Service. Please read more the topic of sequencing mixtures in our Results Interpretation Guide.

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 Guide.

  • Consensus sequence (.fasta file): Polished consensus sequence of the linear/PCR molecule.
  • Consensus sequence (.gbk file): Polished consensus sequence of the linear/PCR molecule, with a feature map and annotations.
  • Molecule map (.html file): An interactive version of the feature map. Note that this map will be depicted as circular, but the bold black bar at position 1 indicates that it is indeed linear.
  • Read length histogram (.png file): Displays the read length distribution of the raw reads produced by your sample. Note that you will typically see a smear of read lengths due to minimal fragmentation during the library prep process.
  • Virtual gel (.png file): Displays the raw read lengths from all samples in the order in a virtual gel format. Note that you will typically see a smear of read lengths due to minimal fragmentation during the library prep process.
  • 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 (.tsv files): Indicates how well the raw reads agree with the consensus sequence at each position.
  • Summary file (.txt file): Indicates the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.) and %. E. coli genomic DNA contamination.
  • 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 linear/PCR 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/PCR Samples

This service requires clonal, linear, 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.

For the purified service, submit the final purified linear/PCR DNA in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing DMSO whenever possible. NOTE: Unpurified samples are more likely to fail and/or you may be charged a purification fee. Submit purified PCR products for best results.

Linear/PCR 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 linear/PCR 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!

This service is intended for linear double-stranded DNA molecules. We recommend performing size verification on full-length linear DNA via gel electrophoresis.

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.

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 linear molecule. Sending mixtures of molecular species is not a supported application and is at your own risk!

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.