Amplicon

We sequence each sample with Oxford Nanopore long reads to very high depth before generating a consensus/assembly using the latest basecalling and polishing software:

• We construct an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including minimal fragmentation of the input linear DNA in a sequence-independent manner
• We sequence the library with a primer-free protocol using the most accurate R10.4.1 flow cells (raw data is delivered in .fastq format).
• We use 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.

As per Oxford Nanopore's specs for the chemistry and flowcells we currently use for Premium PCR sequencing, the consensus accuracy is typically >99.99%.

Results for the Linear/PCR service are typically returned next business day, and results for Premium PCR service are typically returned within 1-2 business days.

This is the average time it takes to return data to customers who are submitting orders from US, UK and EU. Turnaround times for customers in APAC may be slightly longer due to the time required in transit to our lab in Singapore.

Low sample quality can also negatively impact turnaround time and the quality of your results. Please read sample prep instructions carefully to make sure your samples meet input requirements. Due to variability in shipping logistics and sample quality that is outside our control we cannot guarantee turnaround times or sample success rates.

Our Linear/PCR product works in the same way as our Whole Plasmid service: you will receive an annotated assembled consensus of your submitted amplicon. The exact number of reads used to create this assembly varies based our internal quality control metrics, but is typically over a thousand.

The amount of sequencing coverage you will receive for Premium PCR is dependent on which option you choose:

Linear/PCR is intended for a clonal population of molecules. If your species are very similar (e.g. differ by only a few nucleotides), the pipeline will most likely create a single consensus file, with mixed peaks observed in the .ab1 file where there are SNPs and indels.

If your species are sufficiently distinct in size or sequence, the pipeline will generate a single consensus sequence for the molecular species that produces the largest amounts of total sequencing data.

Ultimately, which species ends up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance of each species. Sequencing is considered successful if the pipeline is able to generate a consensus, even if it is not your target.

Re-sequencing mixtures won't change the relative proportions of the species (and hence which species generate a consensus). If you'd like to sequence a known mixture (e.g. barcode or variant libraries), please use our Premium PCR service.

Linear/PCR samples are prepared using the same process as our Whole Plasmid service: the rapid barcoding approach from Oxford Nanopore Technologies. This makes use of a transposome complex to fragment the DNA, simultaneously preparing the ends for the attachment of the sequencing adapter. This is ideal for circular plasmids, as there will be one cut at some random point of the plasmid and the adapter will attach, resulting in full length plasmids. For a linear piece of dsDNA, it still needs to make a cut to attach the adapter and so none of the reads will be full length. For a clonal population of molecules, this doesn't pose a problem, and the fragments are straightforward to reassemble. As you might guess from the name, the rapid approach is lightning fast, and so our Linear/PCR service has the shortest possible turnaround time (one business day).

On the other hand, for a mixed population of molecules, reassembling is going to pose a problem because there is no way of knowing for certain which fragments originally belonged to which molecular species. So our Premium PCR samples are prepared using the native barcoding approach from Oxford Nanopore Technologies. This ligates the sequencing adapter to the ends of the molecules, preserving their full length which makes them extremely easy to tell apart—the single molecule nature of the sequencing platform means that each read corresponds to one specific molecule. This library prep is less rapid, which is why there is a slightly longer turnaround time (one to two business days).

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. 

• 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.
• Virtual gel (.png file, only for Premium PCR): Displays the raw read lengths from all samples in the order in a virtual gel format.
• Reference alignment analysis (comparison-results.tsv file ): A summary of comparisons between samples in the order and the customer-supplied reference sequences for the order, including any mismatches identified.
• 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.
• 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. 

Nanopore sequencing has two known error modes: methylation sites and homopolymers with a length >9. In both cases, in order to prevent this causing any downstream issues, our bioinformatics pipeline clearly labels these errors in light blue:

If you're interested in learning more, see our blog post here for more details.

Assemblers sometimes have difficulty reconstructing the terminal ends of linear DNA, which may result in up to ~25 nucleotides missing from the 3’ and/or 5’ ends of your insert.

For Linear/PCR samples, "failure" means that your sample did not produce data of sufficient quality and quantity for the pipeline to generate a consensus sequence. In order to deliver our extremely fast turnaround times we do not perform extensive pre-sequencing QC of your samples. However, by far the most common reasons are:

• Sample DNA concentration is lower than our specifications
  • The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent spectrophotometry approach.
  • You may see evidence of this failure mode in the low amount of total data reported in the raw read length histogram
• Samples contain a mixture of linear/PCR species, fragmented linear/PCR products, and/or fragmented genomic DNA
  • Because this service includes minimal fragmentation, even successful samples will display a smear of read lengths on the read length histograms. Therefore this failure mode can be difficult to diagnose from the histogram alone, so you may need to rely on other metrics.

The most common reasons for Premium PCR failures overlap heavily with the above. To achieve optimal sequencing results, please follow our recommended sample prep instructions.

Linear/PCR samples are considered to be successfully sequenced if our analysis pipeline generates a consensus sequence.

Premium PCR sample sequencing is considered successful:

• If the sample generated a sufficient number of reads to generate a consensus sequence.
• For cases where our analysis pipeline was unable to generate a consensus sequence (as may occur with highly diverse libraries), if the sample generated at least the following number of reads:
  • Standard - 2,000 reads
  • Big - 4,000 reads
  • Huge - 8,000 reads

This approach tries to strike a balance between returning useful data back to our customers as quickly as possible and trying to provide the target read depth. Due to variability in customer sample quality that is outside our control, we do not guarantee your sample will generate a specific level of read depth, but we do our best to achieve the target.

If your sample failed you may qualify for a re-run. See the FAQ “Does my failed sample qualify for a re-run?” for more information.
 

You are welcome to submit a rerun request for any failed sample through your Order Info page or via the support@plasmidsaurus.com email address. We will evaluate whether you sample quality and quantity permits rerunning the sample (and we may also ask you to provide a reference sequence).

Sample quality checks may require any of the following: 

• Quantify concentration and/ or purity
• Sample clean-up using the Bead Clean Up Protocol ($5 per sample)
• Normalizing concentration

If you have questions about your results, please get in touch at support@plasmidsaurus.com