Premium PCR Sequencing

Sample Type	Category	Size	Concentration	Minimum Volume	Price Per Sample (USD)
Premium PCR	Standard	See Instructions Below		10 μl	$30
	Big	See Instructions Below		20 μl	$60
	Huge	See Instructions Below		40 μl	$120
	Standard with Cleanup	See Instructions Below		10 μl	$35
	Big with Cleanup	See Instructions Below		20 μl	$65
	Huge with Cleanup	See Instructions Below		40 μl	$125

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). In the vast majority of cases, we deliver Premium PCR sequencing results within one business day of receipt of your samples.

Premium PCR 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. Standard Premium PCR typically yields about 3,000 raw reads per sample, Big Premium PCR typically yields about 6,000 raw reads per sample, and Huge Premium PCR typically yields about 12,000 raw reads per sample.
• 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 support@plasmidsaurus.com to ask for up to 3 additional consensus sequences to be generated per sample.

If you need more than 12,000 reads to characterize your molecular mixture or you require >4 consensus sequences generated per sample, 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.
• 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.
• 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 (.tsv file): Indicates how well the raw reads agree with the consensus sequence at each position.
• Summary file (.txt file): Includes (a) the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.) by moles and mass and (b) %. E. coli genomic DNA contamination for the consensus sequence.
• 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 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

• Sample type: Linear double-stranded DNA.
• Size: 100 bp - 25 kb
• Purity:
  • Sample DNA must be purified over a column (e.g. Zymo C & C) or via magnetic beads (e.g. Ampure XP) and free of any primers. Unpurified samples may fail.
  • To save you time, Plasmidsaurus can purify your samples for a small additional fee - simply select the cleanup option.
  • Premium PCR samples are sequenced without primers, so please do not ship any primers with your samples or mixed into your samples.
  • 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.
• Buffer: elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing DMSO.
• Volume: Submit at least 10 μl of each Premium PCR sample in 200 μl strip tubes.
• Concentration: 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 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

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:

Verify Concentration

Verify Quality

Verify Purity

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.