Premium PCR Sequencing

Description of Service

The Premium PCR service is intended for the full-length, fragmentation-free sequencing of linear, double-stranded DNA between 100 bp and 25 kb in length. This service is intended as an alternative to our regular Linear/PCR service (which includes minimal fragmentation during the library prep process).

Premium PCR takes longer to sequence (one week) and is more expensive ($30 per sample) to run than the regular Linear/PCR service, but is ideal in the following scenarios:

  • If your linear DNA sample is NOT clonal, but rather contains a mixture of different molecules (e.g. barcode or variant libraries) that you would like to fully characterize.
  • If you require full-length, end-to-end reads that are sequenced without any fragmentation.
  • If you require a larger number of sequencing reads beyond the typical yield from the regular Linear/PCR service. Premium PCR yields up to 5,000 raw reads per sample.
  • If you require more accurate raw reads than the regular Linear/PCR service, with higher per-base confidence.
  • If you require obtaining ALL raw reads produced by your sample, rather than just the raw reads that align to your consensus as with the regular Linear/PCR service. (Please note that returning ALL raw reads means there is a low level chance of demultiplexing error, so a few reads from your sample might be returned to another customer on the same sequencing run.)

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 including end-ligation for linear DNA. Your input DNA is not fragmented.
  • 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).
  • Aligning the raw sequencing reads against each to generate a high-accuracy linear consensus sequence for the single most abundant molecular species in your sample. If your sample contains multiple molecular species, you may perform your own analyses on the raw reads that are included with your results, or email to ask for additional consensus sequences to be generated.

If you need more than 5,000 reads to characterize your molecular mixture, you can submit multiple aliquots of each sample to the Premium PCR service, or you can submit instead to our Custom Sequencing Service where we can obtain as much data as you specifically require.

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 most abundant molecular species in your sample
  • Consensus sequence (.gbk file): Polished consensus sequence of the most abundant molecular species in your sample, with a feature map and annotations.
  • 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 file): Indicates how well the raw reads agree with the consensus sequence at each position.
  • Raw read sequences (.fastq.gz file): Provides the sequences of all raw reads produced by your sample. Please note that returning all raw reads means there is a small chance of demultiplexing error, so a few reads from your sample might be returned to another customer on the same sequencing run.
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 Premium PCR Samples

Premium PCR sequencing is based on the molarity of your DNA insert, therefore the required concentration (ng/uL) will vary depending on your insert size. Please send your samples at the minimum volume and at the target concentration specified in the table below:

Amplicon Size     DNA Concentration
100 bp 1 ng/μl
200 bp 2 ng/μl
500 bp 5 ng/μl
1000 bp 10 ng/μl
2000 bp 20 ng/μl
5000 bp 50 ng/μl
10000 bp 100 ng/μl
15000 bp 150 ng/μl
20000 bp 200 ng/μl
25000 bp 250 ng/μl

Single-stranded DNA is not currently a supported input 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.

Submit the final purified linear DNA in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing DMSO whenever possible. Unpurified samples may fail and/or you may be charged a purification fee.

Premium 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 your Premium PCR samples at the target concentration and minimum volume specified in the table above. 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

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.