Results Interpretation Guide

Lots of reasons!

• Scientific rigor and peace of mind.
• E. coli and other hosts will go to great lengths to avoid expressing your leaky toxic gene, including modifying your plasmid in unexpected ways that are invisible to targeted Sanger sequencing.
• Plasmid inserts are getting longer and more complex. Instead of multiple Sanger runs or synthesizing a sequencing primer or doing primer walking, sequence the whole plasmid.
• Long reads are ideal for resolving repetitive regions that stymie Sanger sequencing.
• Are you sure your plasmid isn't a dimer? Are you sure there aren't multiple plasmids in your strain? Sanger sequencing won't tell you, and we see it all the time.
• It's neither much more expensive nor slower.

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 linearization of the circular input 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 generate a high-accuracy circular consensus sequence from the raw reads.
• For standard size plasmids, we will also return a set of feature annotations.

Plasmid and circular samples are sequenced WITHOUT primers or amplification. Please do not ship any primers with your samples or mix primers into your samples.

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

This service is intended for a clonal population of molecules. You can send mixtures of molecular species, but since we can't predict the analysis outcome, it's at your own risk.

• If your species are very similar (e.g. differ by only a few nucleotides), the pipeline will most likely create a single .gbk consensus file, with mixed peaks observed in the .ab1 file at SNP/indel locations.
• If your species are sufficiently distinct (e.g. vastly different 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. (Note that concatemer forms such as dimers, trimers, etc. are not considered different molecular species by the pipeline, so you will only receive the monomer consensus sequence by default).

Ultimately, which species ends up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.

Sequencing is considered successful if the pipeline is able to generate any consensus, even if it is not your target. Re-sequencing mixtures won't change the relative proportions of the species (and thus which species generates a consensus), but you can submit multiple aliquots if you need higher overall coverage.

If you'd like to sequence a known mixture (e.g. barcode or variant libraries), please consider submitting instead to our Custom Sequencing Service.

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

The most common error modes for Oxford Nanopore are deletions in homopolymer stretches (especially if longer than 8 bp), errors at the Dam methylation site GATC, and errors at the middle position of the Dcm methylation site CCTGG or CCAGG. These limitations are expected to improve with future updates to ONT sequencing chemistry and basecalling software.

We do not guarantee any specific level of coverage, as the number of raw reads generated can vary substantially depending on sample quality.

Successful samples sent at the required concentration typically yield in the high dozens to hundreds (or thousands!) of raw sequencing reads.

Average coverage is reported in the SAMPLE_summary.tsv file. Coverage over ~20x indicates a very accurate consensus.

The histogram displays the lengths of the raw reads produced by your sample, with read length (bp) on the x-axis and thousands of bases of data collected (kb) at that length on the y-axis. The histogram is therefore weighted by amount of sequencing data produced by different sizes of molecules; for example, two DNA fragments of different lengths that produce the same number of reads will produce different amounts of total data.

The x-axis is automatically scaled to the maximum read length produced by your sample. Before sequencing your plasmids, we linearize them so that we get mostly full-length sequence reads. As a result, the lengths of the raw sequencing reads reflect the lengths of the molecular species in your sample.

Additionally, the histogram color key indicates what fraction of the raw data maps to the consensus sequence:

Ideally, your target plasmid will be the only species in the sample, and we will see one dominant peak in the read length histogram:

If your raw reads contain varying numbers of indels (common for noisy raw reads), this may sometimes cause the read lengths to straddle a bin boundary and artifactually create an appearance of two separate peaks:

More often than you would expect, though, we see multiple peaks corresponding to multiple plasmids, or a peak of a different size than the customer expected:

If you sample contains a mixture, we will return only a single consensus for the molecular species that produces the largest amount of total sequencing data. If you’d like us try generating a consensus for an alternate peak instead, you can email us at support@plasmidsaurus.com to inquire.

Occasionally we see a sample with a dominant peak in addition to an abundance of degraded DNA (genomic and/or plasmid). In some cases the dominant peak may still produce a consensus, if read coverage and accuracy are sufficient:

Sometimes we see a decent number of reads for the sample but there is NO dominant peak, indicating an abundance of degraded DNA (genomic and/or plasmid) from a poor plasmid prep, or that the strain contains no plasmids:

Often, the read count is too low to distinguish any peaks or to generate any consensus:

We see concatemers like this all the time -- they are not a sequencing artifact. Sanger sequencing can't detect them and you won't see them on gel of your digested/linearized plasmid, so you're not used to seeing them, but they turn out to be very common. If you run your sample uncut on a gel with a supercoiled ladder, you will see the concatemer band.

They often seem to be formed in vivo during growth in a RecA+ strain (such as NEB Turbo cells), and are more common when plasmids have large repetitive regions or other complex structures. Even plasmid manufacturers like Addgene observe that concatemers occur frequently, and that only the long-read sequencing technologies like the one we use here Plasmidsaurus (that is, Oxford Nanopore Technologies) can detect them!

Please note that concatemer forms such as dimers, trimers, etc. are not considered different molecular species by the pipeline, so you will only receive the monomer consensus sequence by default, even if other concatemer forms produced more sequencing data.

The .ab1 format has widespread use in Sanger sequencing and normally indicates the intensity of fluorescent nucleotides (A, T, G, C) at each position of the consensus. Since fluorescence is not employed in the Oxford Nanopore Sequencing technology that we use here at Plasmidsaurus, we generate this .ab1 file synthetically using the relative abundance of each nucleotide (A, T, G, C) from the raw reads at each position of the consensus sequence. Because this file type was originally used for Sanger sequencing (which is limited to much shorter read lengths than we get with Oxford Nanopore), the file has a maximum size limit and therefore we must often report sequences in multiple pieces.

This file gives a visual representation of polymorphisms and molecular mixtures present in the sample, and putative insertions can be observed more clearly. An ideal high accuracy basecall will have a sharp, distinct peak of a single color indicating a single nucleotide, whereas a low accuracy basecall will have a less defined or mixed (overlapping) peaks.

For plasmids, "failure" means that your sample did not produce data of sufficient quality and quantity for the pipeline to generate a consensus sequence.

Our low sequencing prices and fast turnaround times do not include extensive QC to determine why your plasmid samples failed (or had low coverage). Although we do not provide definitive reasons for failure, by far the most common reasons are:

• Samples are not prepared at the required DNA concentration.
  The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent.
  You may see evidence of this failure mode in the low amount of total data reported in the raw read length histogram and in the low consensus coverage reported in the SAMPLE_summary.tsv file.
• Samples contain a mixture of plasmid species and/or fragmented genomic DNA or fragmented plasmids.
  You may see evidence of this failure mode in a wide range of read lengths reported in the raw read length histogram.

To achieve optimal sequencing results, please follow our recommended plasmid sample prep instructions, ZeroPrep cell prep instructions, or RCA sample prep instructions

It is relatively rare that we cannot return a consensus sequence, but some rate of failure is unavoidable. You are welcome to submit a rerun request for any failed plasmid samples through your Order Info page (please note that ZeroPrep and RCA Sequencing Services are NOT eligible for reruns). We will evaluate whether your plasmid sample quality and quantity permits rerunning your sample (we may also ask you to provide a reference sequence). We do still charge for failed samples.

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.

Linear/PCR samples are sequenced WITHOUT primers or amplification.Please do not ship any primers with your samples or mix primers into your samples.

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

This service is intended for a clonal population of molecules. You can send mixtures of molecular species, but since we can't predict the analysis outcome, it's at your own risk.

• If your species are very similar (e.g. differ by only a few nucleotides), the pipeline will most likely create a single .gbk consensus file, with mixed peaks observed in the .ab1 file at SNP/indel locations.
• If your species are sufficiently distinct (e.g. vastly different 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. (Note that concatemer forms such as dimers, trimers, etc. are not considered different molecular species by the pipeline, so you will only receive the monomer consensus sequence by default).

Ultimately, which species ends up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.

Sequencing is considered successful if the pipeline is able to generate any consensus, even if it is not your target. Re-sequencing mixtures won't change the relative proportions of the species (and thus which species generates a consensus), but you can submit multiple aliquots if you need higher overall coverage.

If you'd like to sequence a known mixture (e.g. barcode or variant libraries), please consider submitting instead to our Custom Sequencing Service or Premium PCR Service.

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

Depending on the sequence of your sample, the assembler does 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.

The most common error modes for Oxford Nanopore are deletions in homopolymer stretches (especially if longer than 8 bp), errors at the Dam methylation site GATC, and errors at the middle position of the Dcm methylation site CCTGG or CCAGG. These limitations are expected to improve with future updates to ONT sequencing chemistry and basecalling software.

We do not guarantee any specific level of coverage, as the number of raw reads generated can vary substantially depending on sample quality.

Successful samples sent at the required concentration typically yield in the high dozens to hundreds (or thousands!) of raw sequencing reads.

Coverage over ~20x indicates a very accurate consensus.

• Consensus sequence (.fasta file): Provides the polished consensus sequence of the linear/PCR molecule, generated from the raw reads.
• Consensus sequence (.gbk file): Provides the polished consensus sequence of the linear/PCR molecule, generated from the raw reads. Also includes a molecular map and feature annotations from the excellent pLannotate tool from the Barrick Lab:
  McGuffie,M.J. and Barrick,J.E. (2021) pLannotate: engineered plasmid annotation. Nucleic Acids Research DOI: 10.1093/nar/gkab374
• Molecular map (.html file): An interactive version of the molecular 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, thereby providing unique insight into the contents of your samples. 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, resembling what you’d see if you ran the DNA fragments on a gel. 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. See more details about how to interpret your chromatograms below.
• Coverage plot (.png file): Displays the relative sequencing coverage at each position of the consensus sequence. A region with a large gap or a sudden large increase suggests either an assembly issue or a mixture of multiple plasmid species.
• Per-base data (.tsv file): Indicates how well the raw reads agree with the consensus sequence at each position. The list includes the consensus basecalls at each position, along with number of total raw reads aligning at that position and the basecall distributions in the raw reads for that position (A, T, G, C, matches, mismatches, insertions, deletions, etc.).
• Summary file (.txt file): Indicates the % distribution of the various concatemer forms of the consensus sequence.
• 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. Note that any raw reads that do not align to the consensus (e.g. host genomic DNA, lower abundance molecular species) are excluded.
  FASTQ is a sequence format similar to FASTA, with additional Phred quality score information that can displayed graphically by most modern sequence viewers. Since each basecalled position only has one quality score, certain sequence features, such as insertions or deletions, must be inferred from looking at adjacent bases.
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.

The .ab1 format has widespread use in Sanger sequencing and normally indicates the intensity of fluorescent nucleotides (A, T, G, C) at each position of the consensus. Since fluorescence is not employed in the Oxford Nanopore Sequencing technology that we use here at Plasmidsaurus, we generate this .ab1 file synthetically using the relative abundance of each nucleotide (A, T, G, C) from the raw reads at each position of the consensus sequence. Because this file type was originally used for Sanger sequencing (which is limited to much shorter read lengths than we get with Oxford Nanopore), the file has a maximum size limit and therefore we must often report sequences in multiple pieces.

This file gives a visual representation of polymorphisms and molecular mixtures present in the sample, and putative insertions can be observed more clearly. An ideal high accuracy basecall will have a sharp, distinct peak of a single color indicating a single nucleotide, whereas a low accuracy basecall will have a less defined or mixed (overlapping) peaks.

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.

Our low sequencing prices and fast turnaround times do not include extensive QC to determine why your linear/PCR samples failed (or had low coverage). Although we do not provide definitive reasons for failure, by far the most common reasons are:

• You submitted to the "unpurified" service.
  Unpurified DNA (with DNA still intermixed with the original reaction reagents) is more likely to fail. We recommend performing cleanup and submitting the "purified" service next time.
• Samples are not prepared at required DNA concentration.
  The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent.
  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.

To achieve optimal sequencing results, please follow our recommended linear/PCR sample prep instructions.

It is relatively rare that we cannot return a consensus sequence, but some rate of failure is unavoidable. You are welcome to submit a rerun request for any failed samples through your Order Info page. We will evaluate whether your sample quality and quantity permits rerunning your sample (we may also ask you to provide a reference sequence). We do still charge for failed samples.

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 end-ligation of the linear input DNA. Your DNA is not fragmented.
• 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 generate a high-accuracy linear consensus sequence from the raw reads for the most abundant molecular species.

Premium PCR samples are sequenced WITHOUT primers or amplification.Please do not ship any primers with your samples or mix primers into your samples.

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

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 arger 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, for DNA up to 25 kb in length.
• 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.)

Yes! Just note that by default, we will generate only one 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 additional consensus sequences to be generated.

If you need more than 5,000 reads to fully 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.

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%. The raw reads from this service are also more accurate than the raw reads from the regular Linear/PCR Service, with higher per-base confidence.

The most common error modes for Oxford Nanopore are deletions in homopolymer stretches (especially if longer than 8 bp), errors at the Dam methylation site GATC, and errors at the middle position of the Dcm methylation site CCTGG or CCAGG. These limitations are expected to improve with future updates to ONT sequencing chemistry and basecalling software.

For samples sent at the correct concentration, we typically collect 3,000-5,000 raw sequencing reads.

• Consensus sequence (.fasta file): Provides the polished consensus sequence of the most abundant molecular species in your sample.
• Consensus sequence (.gbk file): Provides the polished consensus sequence of the most abundant molecular species in your sample, generated from the raw reads. We also return a molecular map and feature annotations from the excellent pLannotate tool from the Barrick Lab:
  McGuffie,M.J. and Barrick,J.E. (2021) pLannotate: engineered plasmid annotation. Nucleic Acids Research DOI: 10.1093/nar/gkab374
• Molecular map (.html file): An interactive version of the molecular map.
• Read length histogram (.png file): Displays the read length distribution of the raw reads produced by your sample, thereby providing unique insight into the contents of your samples. .
• Virtual gel (.png file): Displays the raw read lengths from all samples in the order in a virtual gel format, resembling what you’d see if you ran the DNA fragments on a gel.
• 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. See more details about how to interpret your chromatograms below.
• Coverage plot (.png file): Displays the relative sequencing coverage at each position of the consensus sequence. A region with a large gap or a sudden large increase suggests either an assembly issue or a mixture of multiple plasmid species.
• Per-base data (.txt and .tsv files): Includes 3 sub-files for each sample:
• SAMPLE.tsv: Indicates how well the raw reads agree with the consensus sequence at each position. The list includes the consensus basecalls at each position, along with number of total raw reads aligning at that position and the basecall distributions in the raw reads for that position (A, T, G, C, matches, mismatches, insertions, deletions, etc.).
• SAMPLE_multimer_analysis.txt: Indicates the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.).
• SAMPLE_summary.tsv: Indicates the length, average coverage, relative composition (by moles and mass), total reads, total bases, and %. E. coli genomic DNA contamination for the consensus sequence.
• Raw read sequences (.fastq.gz file): Provides the sequences of ALL individual raw reads produced by your sample. 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.
  FASTQ is a sequence format similar to FASTA, with additional Phred quality score information that can displayed graphically by most modern sequence viewers. Since each basecalled position only has one quality score, certain sequence features, such as insertions or deletions, must be inferred from looking at adjacent bases.

The .ab1 format has widespread use in Sanger sequencing and normally indicates the intensity of fluorescent nucleotides (A, T, G, C) at each position of the consensus. Since fluorescence is not employed in the Oxford Nanopore Sequencing technology that we use here at Plasmidsaurus, we generate this .ab1 file synthetically using the relative abundance of each nucleotide (A, T, G, C) from the raw reads at each position of the consensus sequence. Because this file type was originally used for Sanger sequencing (which is limited to much shorter read lengths than we get with Oxford Nanopore), the file has a maximum size limit and therefore we must often report sequences in multiple pieces.

This file gives a visual representation of polymorphisms and molecular mixtures present in the sample, and putative insertions can be observed more clearly. An ideal high accuracy basecall will have a sharp, distinct peak of a single color indicating a single nucleotide, whereas a low accuracy basecall will have a less defined or mixed (overlapping) peaks.

For Premium PCR samples, "failure" means that your sample did not produce data of sufficient quality and quantity for the pipeline to generate any consensus sequence from your mixture.

Our low sequencing prices and fast turnaround times do not include extensive QC to determine why your Premium PCR samples failed (or had low coverage). Although we do not provide definitive reasons for failure, by far the most common reasons are:

• You submitted unpurified DNA.
  Unpurified samples (with DNA still intermixed with the original reaction reagents) are more likely to fail and/or you may be charged a purification fee.
• Samples are not prepared at the required DNA concentration. The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent. You may see evidence of this failure mode in the low amount of total data reported in the read length histogram or in the small number of raw .fastq reads received.
• Samples contain a mixture of DNA species, but none of them obtained sufficient coverage on its own to produce a consensus.

To achieve optimal sequencing results, please follow our recommended Premium PCR sample prep instructions.

• If your purified DNA sample failed to produce a consensus, but you received MORE THAN 2,000 full-length raw reads, this suggests that your sample contains a diverse mixture of products and/or the DNA is fragmented. You can email us at support@plasmidsaurus.com to request assembly of a different molecule in the mixture (we may also ask you to provide a reference sequence).
• If your purified DNA sample failed to produce a consensus and you received LESS THAN 2,000 full-length raw reads, you are welcome to submit a rerun request through your Order Info page. We will evaluate whether your sample quality and quantity permits rerunning your sample (we may also ask you to provide a reference sequence).