Frequently Asked Questions


General


Sample type Category Size Concentration Minimum volume Price per sample Order now
Plasmid Standard 2.5 - 25 kb 30 ng/uL ≥10 uL $15 or 1 dinocoin Order
Big 25 - 125 kb 50 ng/uL ≥20 uL $30 or 2 dinocoins Order
Huge 125 - 300 kb 50 ng/uL ≥40 uL $60 or 4 dinocoins Order
Linear/Amplicon Standard 600 bp - 25 kb 30 ng/uL ≥10 uL $15 or 1 dinocoin Order
Big 25 - 125 kb 50 ng/uL ≥20 uL $30 or 2 dinocoins Order
Bacterial Genome Standard up to 7 Mb 50 ng/uL ≥20 uL $90 or 6 dinocoins Order
Big 7 - 12 Mb 50 ng/uL ≥20 uL $105 or 7 dinocoins Order
Standard with extraction up to 7 Mb Send a bacterial pellet in DNA/RNA Shield $105 or 7 dinocoins Order
Big with extraction 7 - 12 Mb Send a bacterial pellet in DNA/RNA Shield $120 or 8 dinocoins Order
Hybrid ONT + Illumina up to 7 Mb 50 ng/uL ≥20 uL $165 or 11 dinocoins Order
Big Hybrid ONT + Illumina 7 - 12 Mb 50 ng/uL ≥20 uL $195 or 13 dinocoins Order
Hybrid with extraction up to 7 Mb Send a bacterial pellet in DNA/RNA Shield $180 or 12 dinocoins Order
Big hybrid with extraction 7 - 12 Mb Send a bacterial pellet in DNA/RNA Shield $210 or 14 dinocoins Order
Pre-paid credits Dinocoins $15 Purchase
The standard plasmid service is optimized for plasmids between 2.5 kb and 25 kb. We charge more for larger plasmids since they take extra sequencing and don't multiplex well with small plasmids.

We will attempt to assemble your plasmid/cosmid/BAC using a bioinformatic pipeline tuned to be generally effective, but note that if your sequence is repetitive or otherwise challenging, we aren't able to give it the personalized attention needed for an optimal assembly. You might want to download the raw reads and try assembling it yourself.

Also note that as plasmids get large, copy numbers get low, and purification becomes difficult. We recommend doing extra QC before you submit to make sure your prep is high quality.
  • plasmids:
    • 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.
    • Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data is >99% accurate and is delivered in .fastq format).
    • Align the raw reads against each other to generate a high-accuracy circular consensus sequence and a set of gene annotations.
  • linear/amplicons:
    • Construct 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.
    • Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data is >99% accurate and is delivered in .fastq format).
    • Re-assemble the raw reads and align them against each other to generate a high-accuracy linear consensus sequence.
  • bacterial gDNA:
    • Construct an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including minimal fragmentation of the genomic input DNA in a sequence independent-manner.
    • Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data is >99% accurate and is delivered in .fastq format).
    • Produce a high-accuracy genome assembly (delivered in .fasta format) with Flye for assembly and medaka for polishing.
    • Produce a set of bacterial genome annotations with Bakta (delivered in various file formats).

In all cases, samples are sequenced without primers or amplification. Please do not ship any primers with your samples or mix primers into your 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 strongly recommend using Qubit or equivalent to measure DNA concentration. Nanodrop is NOT ADEQUATE due to low accuracy.

No. Nanodrop is NOT ADEQUATE for DNA quantification due to low accuracy. The extent to which Nanodrop is off varies widely depending on the specific composition of your sample, so there is no standard way to “adjust” Nanodrop values. Sending samples at too high OR too low concentration based on Nanodrop values may adversely affect the library prep and/or sequencing reactions, possibly resulting in sequencing failure.

For proper citation, use the following phrase: ‘Type of Service was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation.’ Please change Type of Service accordingly to Whole plasmid sequencing, Bacterial genome sequencing or Linear/Amplicon sequencing.

Submitting Orders


Please register for an account. When you log in, you will be directed to your Dashboard and will see options on the screen to submit your order.

You can cancel an order from the order details page, reached via the magnifying glass button on your Dashboard.

We will accept any number of sample(s).

Yes, absolutely!

Shipping


  • For US customers:

    Our Dropboxes and the free & reduced cost UPS labels generated during order submission both provide overnight shipping within the US to our Louisville lab location, and both provide next-morning results for whole plasmid and linear/amplicon sequencing. We recommend that US customers use either a Dropbox or a plasmidsaurus-generated UPS label whenever possible (rather than self-shipping).

  • For international customers:

    International Dropbox shipments are usually delivered to us within 2-3 days and we provide results on the same morning of receipt. If you do not have a local dropbox, you can self-ship via any carrier to our Louisville lab location:

    plasmidsaurus
    4743 Poplar Level Road
    Louisville, KY 40213

    Please ensure any required forms are included with your shipment.

    NOTE ABOUT DROPBOXES: The specific day that each dropbox ships out varies by site, and may not be every weekday for your local dropbox. Please check the dropbox list to find the shipping day(s) for your local dropbox! To ship your samples on a different day, please refer to "Alternative Shipping Options" in the detailed shipping table. Contact us at support@plasmidsaurus.com if you’re interested in hosting a new dropbox at your location!

  • For US customers:

    The free & reduced cost UPS labels generated during order submission provide overnight shipping within the US to our Eugene lab location and provide results within 1 business day for bacterial genome sequencing. We recommend that US customers use the plasmidsaurus-generated UPS label whenever possible (rather than using a Dropbox or self-shipping).

  • For international customers:

    Free & reduced cost UPS labels are only available to US customers at this time, therefore for international customers, using a Dropbox will provide fastest turnaround time. International Dropbox shipments are usually delivered to us within 2-3 days and we provide results within 1 business day of receipt. If you do not have a local dropbox, self-ship via any carrier to our Eugene lab location:

    plasmidsaurus
    1850 Millrace Drive, Suite 200
    Eugene, OR 97403

    Please ensure any required forms are included with your shipment.

    NOTE ABOUT DROPBOXES: The specific day that each dropbox ships out varies by site, and may not be every weekday for your local dropbox. Please check the dropbox list to find the shipping day(s) for your local dropbox! To ship your samples on a different day, please refer to "Alternative Shipping Options" in the detailed shipping table. Contact us at support@plasmidsaurus.com if you’re interested in hosting a new dropbox at your location!

If you placed an order of 8+ plasmids/amplicons, 4+ Bigs, 2+ Huges/Bacteria or larger and requested a free domestic UPS shipping label it will be automatically downloaded at the order confirmation screen to your browser’s download folder.

Please ensure any required forms are included with your shipment. Here is some general advice:

  • We recommend shipping with FedEx, as it has worked best for international shipments in our experience.
  • Make sure all forms are fully completed. Incomplete and/or missing forms will likely cause delays and other issues.
  • Keep an eye out for any communication or information requests from FedEx in case of issues. It is your responsibility to provide any information they request.
  • For further international shipping queries, use FedEx International Shipping Assist.
Recommended forms to include with international plasmid and/or microbial genome DNA shipments:
  • Commercial Shipping Invoice (Download Template)
    For best results, make sure to include the shipper's identity, shipper's address, and the quantity of items within the shipment.
  • Manufacturers Identification (MID) code. The manufacturer will be whoever generated the plasmids, in most cases this will be yourself unless the plasmids were sourced from another supplier.
  • Detailed Description (Download Template)
    A detailed description of the material. For example: "The enclosed material is plasmid DNA which does not contain a vector that is pathogenic to plant, avian, or livestock species, and does not contain any other materials above Biosafety Level 1 (BSL1). The material is not derived from animal cultures and contains no animal products. The plasmids are diluted in water or Tris buffer. Plasmids are produced by microbial fermentation of laboratory strains of E. coli and yeast and isolated using a plasmid purification kit. The end use of the material is DNA sequencing. This material does not contain any infectious biological agent. Samples are isolated from non-infectious laboratory strains, and chemically treated during the purification process with a lysis buffer that destroys living cells. As per USDA Guidelines 1110, 1114, and 1116. Harmonized Tariff Number: 2934999000."
  • Toxic Substances Control Act (TSCA) Certificate (Download Template)
    We recommend writing something like "DNA for research purposes only" in the Product Description section.
    TSCA Certificate is not needed if you are shipping samples to our London lab.

We update your Dashboard with the received date immediately upon opening your package. If you do not see a received date in your Dashboard, then we have not yet received your samples, although we may be still in the process of opening packages that have been delivered. Keep an eye on your Dashboard for updates.

No, your package will be held by the carrier and delivered to us on Monday. Plasmids and high-quality bacterial genomic DNA are quite stable, so the wait isn't a problem.

Please Check our Dropbox Map or the Dropbox List to find a plasmidsaurus dropbox near you. You can find information about dropbox pick-up times and locations on the map or from the list.

If there isn't one at your institution yet, we are interested in setting one up! Contact us at support@plasmidsaurus.com if you are willing to host one.

First go to the Dropbox List on our website and search for the dropbox that was used for shipping us your samples. Once there, click the “UPS” button to the left of the dropbox entry under the “Tracking” column. This will take you to a new page with a list of tracking numbers and the date the corresponding shipment should be sent out. Just click the tracking number for the shipment from the week your samples were submitted and it will take you to the UPS tracking page for that shipment.

You don’t need to notify us in order to have your samples collected from our dropboxes. Any and all orders that are dropped-off in one of our dropboxes will be packaged and shipped to us on the listed cut-off day/time posted on the dropbox and on our website.

Payments


Look us up under the company name SNPsaurus

Never any extra fees! The prices listed in the table above are what we will charge you.

Please generate your own quote using our quote tool!

You can pay by credit card, purchase order (PO), or dinocoins at the time of submission. You can also elect to have a credit card payment link emailed to a purchasing agent at your institution. If you need to make alternate payment arrangements, email us at remit@snpsaurus.com.

If you will be placing multiple orders using purchase orders, please consider using a blanket PO (also called a standing PO) or dinocoins. This simplifies the ordering process for you and the invoicing process for us. Blanket POs can be used by individuals or a lab group.
Get a quote for the amount you think you will use over the next few months and your purchasing department will issue a PO for that amount. Check first - not all organizations allow blanket POs. You would use that PO number every time you order and we invoice the blanket PO every month or so.

Tired of dealing with credit cards and purchase orders? Got a grant that's expiring? Load up on dinocoins!

Dinocoins are pre-paid credits that can be used to pay for sequencing. When you have Dinocoins in your account, you can “apply Dinocoins” during checkout to pay for an order.

Dinocoins never expire and hold their dollar value.

Dinocoins cannot currently be used to partially pay for orders -- it's all or none.

You can pay for Dinocoins by credit card, payment link or PO.

If you order using a payment link, the Dinocoins will not be available in your account until the link is paid.
If you use a PO, you must purchase Dinocoins from your Dashboard on the plasmidsaurus website (see the last row of the order table) to populate your account with the Dinocoins. You can see all your transactions and the number of Dinocoins you have left on your Dashboard so it is easy to monitor usage.

For credit card orders, invoices are issued at the time of placing an order to the email address associated with the submission account. Credit card invoices can also be downloaded from your Dashboard.
For POs, invoices are generally issued on a monthly basis to the billing address listed on the PO.

Whole Plasmid Sequencing Service


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.

In the vast majority of cases, we deliver plasmid sequencing results:

  • the next morning for US customers who ship overnight using our Dropbox or UPS label generated during order submission.
  • within 2-3 business days for international customers who ship overnight.
  • within one business day of receipt of samples for customers who choose alternative shipping options.

Please see How to ship whole plasmid and linear/amplicon sequencing samples to get the fastest turnaround time?

Our service for plasmids 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 .fasta consensus file with low confidence positions at SNP/indel locations. You can view those locations in your provided stats.csv and .fastq files.
  • If your species are sufficiently distinct (e.g. vastly different in size or sequence), the pipeline will first attempt to make a .fasta consensus file from the highest abundance species. It will also attempt to make a consensus of other species with read counts that are >20% of that of the most abundant species. Ultimately, which species end 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, but you can submit multiple aliquots if you need higher total coverage.

If the pipeline does not produce a consensus for your target, you can download the raw reads from your Dashboard and bin them yourself.

If you are interested in sequencing a known mixture (e.g. barcode or variant libraries), please refer to the section below on Custom Sequencing Service.

As per Oxford Nanopore’s specs for the chemistry and flowcells we currently use for plasmid sequencing, the raw read accuracy is >99%.
Deeper coverage — meaning more reads from which to build a consensus — generally increases the accuracy of results.

The most common error modes for Oxford Nanopore are deletions in homopolymer stretches and errors at the Dam methylation site GATC.
This limitation is expected to improve with future updates to their sequencing chemistry and basecalling software.

We do not guarantee any specific level of coverage. 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. Final coverage of the consensus depends on how many of the raw sequencing reads are full-length plasmids and whether any degraded plasmid reads can be aligned to the full-length consensus.
Average coverage is reported in the header line of the fasta file. Coverage over ~20x indicates a very accurate consensus.

  • .fasta file (for consensus data): We return a polished consensus sequence of each plasmid in fasta format.
  • [OPTIONAL] .fastq file (for raw reads): If you like, you can elect during the order process to receive the raw fastq sequencing reads. You can also download the raw reads anytime from your Dashboard.
  • .svg file (for raw reads): We return raw read length histograms for each plasmid, which provide unique insight into the contents of your samples. See details on interpreting your histograms.
  • .html pLannotate map (for consensus data): We return a plasmid map for each sample, generated with the excellent pLannotate tool from the Barrick Lab.
  • .gbk GenBank file (for consensus data): We return the same pLannotate map in GenBank file format.

pLannotate image

McGuffie,M.J. and Barrick,J.E. (2021) pLannotate: engineered plasmid annotation. Nucleic Acids Research DOI: 10.1093/nar/gkab374

Finally, we return two files that show how confident we are in our basecall at each position of our consensus sequence:

  • .csv file (comparing raw reads to consensus data): We align the raw reads to the consensus results and return a stats.csv file that lists how well the raw reads agree at each position.
  • .fastq file (comparing raw reads to consensus data): We create this .fastq file from the stats.csv file to visualize our consensus confidence. It contains the consensus base call and a “consensus score” (an internal metric of our confidence for the consensus basecall) for each position. This .fastq file can be viewed in software such as SnapGene Viewer to quickly identify low-confidence positions.

    sample .fastq file

    Shorter bars correspond to lower confidence positions.
    • IMPORTANT: Our "consensus score" is measured on the same Phred scale as standard Q-scores and is reported in the standard range of 1 to 42. This score does not correlate linearly with the percent of raw reads agreeing with the consensus. It is weighted by the number of raw reads available, ONT's machine learning algorithms for basecalling, and other statistical considerations. For example, a "consensus score" lower than 20 corresponds to a low-confidence position where under 70% of the raw reads match the consensus.
    • IMPORTANT: This .fastq file for the consensus is DISTINCT from the .fastq files for the raw read data. This consensus .fastq file will be delivered with your standard sequencing results, whereas the .fastq file for the raw read data must be requested during order submission or downloaded separately from the Dashboard.

Note we do not return chromatograms (.ab1 files) because we do not do Sanger sequencing. The confidence files described above provide similar information.

The histogram displays raw read lengths between 1-25kb (for standard plasmids) and is subdivided into 100 bins. Before sequencing your plasmids, we linearize them so that we get mostly full-length sequence reads. As a result, the lengths of our raw sequencing reads reflect the lengths of the molecular species in your sample.

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

histo-one-species

A dominant peak (~4,800 bp in this case) typically suggests a clean prep with a single plasmid.

(Please note that even a single apparent peak MAY contain multiple plasmids of the same size, or multiple plasmids of different lengths that happen fall into the same histogram bin. Sequences that are very similar are assumed by the analysis pipeline to be variations of a single species and it will attempt to make a single consensus (with potentially low confidence positions reported in the stats.txt file); if the sequences are very distinct, it will attempt to produce multiple consensus sequences.)

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:

histo-double-peak

These reads most likely all come from a single plasmid (~2,500 bp in this case), but varying numbers of insertion and deletion sequencing errors result in different lengths that cause them to straddle a bin boundary.

(Please note that a peak straddling a bin boundary MAY contain multiple plasmids of the same size, or multiple plasmids of different lengths that happen fall into two adjacent histogram bins. Sequences that are very similar are assumed by the analysis pipeline to be variations of a single species and it will attempt to make a single consensus (with potentially low confidence positions reported in the stats.txt file); if the sequences are very distinct, it will attempt to produce multiple consensus sequences.)

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:

histo-multi-species-1

Uh oh! Good thing you did whole plasmid sequencing, Sanger sequencing might not have shown you all these plasmid species!

histo-multi-species-2

This sample contains 3 unique plasmid species, only 2 of which (~8,600 bp and ~4,200bp bp -- corresponding to the target plasmid and the empty vector, respectively) yielded enough coverage to produce a consensus.

(The analysis pipeline will provide consensus sequences for any plasmids in the mixture that obtain enough coverage and are at least 20% the height of the max peak in the sample. If you observe a histogram peak here that does not have an accompanying consensus delivered in your data, then coverage of that species was too low to assemble or that peak was actually a mixture of plasmid species. If sequencing fails entirely due to low coverage for all species in the mixture, you will not receive any consensus sequences, but you may find some useful information in the raw reads.)

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:

histo-one-with-degradation

This sample produced a consensus for the ~14,000 bp peak, with the degraded plasmid fragments contributing to its coverage.

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:

histo-no-peak

No dominant peak was observed in this sample, despite high read count. No consensus was generated.

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

histo-low-read-count

If read count is too low, usually it is because samples are not prepared at required DNA concentration.
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, you will see the dimer band. They often seem to be formed in vivo during growth in a RecA+ strain. Note that we don't return stats files for dimers because the raw reads don't align unambiguously to the consensus.

For plasmids, “failure” refers to the failure of your sample to produce a consensus sequence with at least 10x coverage.

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

  • 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 low read counts reported in the raw read length histogram. (Please note that if you decide to evaluate the data from such samples with low read numbers, noise may exceed true sequencing signal, so we advise caution.)
  • 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 sample prep and QC steps.

It is relatively rare that we cannot return a plasmid sequence, but some rate of failure is unavoidable. We may attempt to re-sequence failed samples if your sample quality and quantity permits (with follow-up results delivered in 2-3 business days). If the sample fails a second time, we will conclude that something about the sample makes it unsequenceable. Check out our Sequencing Workflow. Unfortunately we must still charge for failed samples, since we spend more time and resources on them than we do on successes.

Linear/Amplicon Sequencing Service


In the vast majority of cases, we deliver linear/amplicon sequencing results:

  • the next morning for US customers who ship overnight using our Dropbox or UPS label generated during order submission.
  • within 2-3 business days for international customers who ship overnight.
  • within one business day of receipt of samples for customers who choose alternative shipping options.

Please see How to ship whole plasmid and linear/amplicon sequencing samples to get the fastest turnaround time?

Our service for linear/amplicons 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 .fasta consensus file with low confidence positions at SNP/indel locations. You can view those locations in your provided stats.csv and .fastq files.
  • If your species are sufficiently distinct (e.g. vastly different in size or sequence), the pipeline will first attempt to make a .fasta consensus file from the highest abundance species. It will also attempt to make a consensus of other species with read counts that are >20% of that of the most abundant species. Ultimately, which species end 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, but you can submit multiple aliquots if you need higher total coverage.

If the pipeline does not produce a consensus for your target, you can download the raw reads from your Dashboard and bin them yourself.

If you are interested in sequencing a known mixture (e.g. barcode or variant libraries), please refer to the section below on our Custom Sequencing Service.

As per Oxford Nanopore’s specs for the chemistry and flowcells we currently use for linear/amplicon sequencing, the raw read accuracy is >99%.
Deeper coverage — meaning more reads from which to build a consensus — generally increases the accuracy of results.

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. If you observe this happening with your samples, you may download the raw reads from your Dashboard and reconstruct the ends with your preferred method.

The most common error modes for Oxford Nanopore are deletions in homopolymer stretches and errors at the middle position of the Dcm methylation site CCTGG or CCAGG.
This limitation is expected to improve with future updates to their sequencing chemistry and basecalling software.

We do not guarantee any specific level of coverage. 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 header line of the fasta file. Coverage over ~20x indicates a very accurate consensus.

  • .fasta file (for consensus data): We return a polished consensus sequence in fasta format.
  • [OPTIONAL] .fastq file (for raw reads): If you like, you can now also elect during the order process to receive the raw fastq sequencing reads. You can also download the raw reads anytime from your Dashboard.

Finally, we return two files that show how confident we are in our basecall at each position of our consensus sequence:

  • .csv file (comparing raw reads to consensus data): We align the raw reads to the consensus results and return a stats.csv file that lists how well the raw reads agree at each position.
  • .fastq file (comparing raw reads to consensus data): We create this .fastq file from the stats.csv file to visualize our consensus confidence. It contains the consensus base call and a “consensus score” (an internal metric of our confidence for the consensus basecall) for each position. This .fastq file can be viewed in software such as SnapGene Viewer to quickly identify low-confidence positions.

    sample .fastq file

    Shorter bars correspond to lower confidence positions.
    • IMPORTANT: Our "consensus score" is measured on the same Phred scale as standard Q-scores and is reported in the standard range of 1 to 42. This score does not correlate linearly with the percent of raw reads agreeing with the consensus. It is weighted by the number of raw reads available, ONT's machine learning algorithms for basecalling, and other statistical considerations. For example, a "consensus score" lower than 20 corresponds to a low-confidence position where under 70% of the raw reads match the consensus.
    • IMPORTANT: This .fastq file for the consensus is DISTINCT from the .fastq files for the raw read data. This consensus .fastq file will be delivered with your standard sequencing results, whereas the .fastq file for the raw read data must be requested during order submission or downloaded separetely from the Dashboard.

We do not return chromatograms (.ab1 files) because we do not do Sanger sequencing. The confidence files described above provide similar information.

We do not return histograms for linear samples because these samples are fragmented during library prep, thus a clonal peak is not produced on the histogram and it is typically not informative.

For linear/amplicon, “failure” refers to the failure of your sample to produce a consensus sequence with at least 10x coverage.

Our low sequencing prices and fast turnaround times do not include extensive QC to determine why samples fail. Although we cannot provide definitive reasons for failure, by far the most common reasons are:

  • 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.
  • Samples contain a mixture of linear/amplicon species and/or fragmented genomic DNA

To achieve optimal sequencing results, please follow our recommended sample prep and QC steps.

It is relatively rare that we cannot return a linear/amplicon sequence, but some rate of failure is unavoidable. We may attempt to re-sequence failed samples if your sample quality and quantity permits (with follow-up results delivered in 2-3 business days). If the sample fails a second time, we will conclude that something about the sample makes it unsequenceable. Check out our Sequencing Workflow. Unfortunately we must still charge for failed samples, since we spend more time and resources on them than we do on successes.

Bacterial Genome Sequencing Service


In the vast majority of cases, we deliver bacterial genome sequencing results within 1-2 business days of receipt of your samples. When our genomic DNA extraction option is selected, we deliver bacterial genome sequencing results within 1 week of receipt of your samples. If you select the NEW Hybrid sequencing option, we deliver hybrid results with 6-8 business days (or 8-10 business days if you include the extraction option).

As per Oxford Nanopore’s specs for the chemistry and flowcells we currently use for bacterial genome sequencing, the raw read accuracy is >99%.
The accuracy of the final assembly varies depending on coverage and data quality.
Deeper coverage — meaning more reads from which to build a consensus — generally increases the accuracy of results.

Successful sequencing is defined by achieving at least one of the following deliverables:

  • A high-quality genome assembly
  • Target amount of raw data
    • 210 Mb of raw sequencing data for the “standard” service (i.e. 30x genome coverage of a single 7 Mb genome)
    • 360 Mb of raw sequencing data for the “big” service (i.e. 30x genome coverage of a single 12 Mb genome)
If you select the NEW Hybrid sequencing option, we target the same amount of ONT data listed above, PLUS an equal amount of Illumina short-read data:
  • Target amount of raw Illumina data
    • 210 Mb of raw Illumina data for the “standard” service (i.e. 30x genome coverage of a single 7 Mb genome)
    • 360 Mb of raw Illumina data for the “big” service (i.e. 30x genome coverage of a single 12 Mb genome)

However, our ability to deliver these target outputs is directly dependent on the quantity, quality, and purity of the gDNA that is sent to us. We do not guarantee any specific output.

1. Remove the bottom 5% worst fastq reads via Filtlong v0.2.1 (default parameters)
2. Downsample the reads to 250 Mb via Filtlong to create a rough sketch of the assembly with Miniasm v0.3
3. Using information acquired from the Miniasm assembly, re-downsample the reads to ~100x coverage (do nothing if there isn't at least 100x coverage) with heavy weight applied to removing low quality reads (helps small plasmids stick around)
4. Run a Flye v2.9.1 assembly with parameters selected for high quality ONT reads
5. Polish Flye assembly via Medaka v1.8.0 using the reads generated in step 3
6. Run several analyses:

  • annotation
  • contig analysis
  • genome completeness and contamination
  • species / plasmid identification

If you select the NEW Hybrid sequencing option, we target the same deliverables listed above for your ONT-only assembly, PLUS we run the following additional step:

7. Polish the ONT .fna assembly with Illumina .fastq reads using Polypolish v0.5.0, which yields a new .fasta polished hybrid assembly file

  • FASTA files:
    • .fna (contig nucleotide sequences) = polished consensus sequence of the genome
    • .faa (protein amino acid sequences)
    • .ffn (gene nucleotide sequences)
  • GenBank files:
    • .gbff (annotated contig sequences) = polished and annotated consensus sequence of the genome
  • FASTQ file:
    • .fastq.gz = a compressed file of all the raw .fastq sequencing reads
  • Report:
    • .html = An analytical report of the key metrics for the assembly (including completeness of the assembly based on CheckM and general species identification of the contigs)
  • Various other Bakta annotation files

If you select the NEW Hybrid sequencing option, we deliver the same files listed above for your ONT-only assembly, PLUS we deliver the following additional file:

  • FASTA file:
    • .fasta polished hybrid assembly file, made by polishing the .fna ONT assembly with Illumina .fastq sequence reads

If we are not able to achieve at least one of the target deliverables, then we will repeat sequencing as per our bacterial repeat policy.

Even when a high-quality assembly cannot be generated, we still provide the raw data and the report, and you may also still receive some of the other file types.

Although we do not provide definitive reasons on why each specific sample may need to be repeated, by far the most common reasons are:

  • Samples are not prepared at required DNA concentration of 50 ng/uL.
    The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent fluorometric assay.
  • Samples contain fragmented/degraded gDNA.
    At least 50% of the DNA should be above 15kb in length, and samples should be handled with utmost care:
    • Pipetting with wide-bore tips
    • Minimal freeze/thaw cycles
    • No vortexing
    • No extreme temperature/pH
    • No intercalating dyes
    • No UV radiation
    • Not over-dried
  • Samples contain inhibitors, such as:
    • RNA
    • Denaturants (guanidinium salts, phenol, etc.)
    • Detergents (SDS, Triton-X100, etc.)
    • Residual contaminants from the organism/tissue (heme, humic acid, polyphenols, polysaccharides, lipids, etc.)
    • Insoluble, colored, or cloudy material
    • Other inhibitors (EDTA, etc.)
  • The DNA you sent is not from a bacterial isolate.
    This service is intended for a clonal population (single species) of bacteria. If your sample contains a mixture of different bacterial species, it may fail to produce an assembly. Please refer to our FAQ on sequencing mixtures for more information.

To increase chances of successful sequencing on the first attempt, please adhere closely to our sample prep guidelines.

If we are not able to achieve at least one of the target deliverables on the 1st sequencing attempt, we will evaluate the results of the initial sequencing attempt to determine whether additional sequencing may produce a more successful outcome, and if so we will repeat the sequencing (with possible protocol adjustments) at no additional charge. We will also combine the data from the two runs together to increase chances of success on the repeat.

If we are not able to achieve at least one of the target deliverables on the 2nd attempt, we will not perform further repeats.

If you wish to sequence the sample again, please prepare new samples that meet all the QC requirements before submitting a new sequencing request.

This service is intended for a clonal population (single species) of bacteria. You can send mixtures of different bacterial species for sequencing, but since we can't predict the assembly outcome, it's at your own risk.

The total amount of raw data obtained for your sample will be divided up between however many species are present in your sample, thereby reducing each species’ own genome coverage and possibly inhibiting assembly of particular species in the sample. Re-sequencing mixtures won't change the relative proportions of the species, but you can submit multiple aliquots if you need higher total coverage. Ultimately, which species end up producing an assembly will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.

If your gDNA extract also contains native plasmid DNA, then yes, you will probably receive some sequencing reads for those plasmids. Input DNA fragments < 3kb are typically omitted during the sequencing process, but otherwise we do not filter out small plasmid-sized reads during assembly, so it is likely that they will also produce their own plasmid assemblies in addition to the gDNA assembly. Ultimately, which types of DNA in your sample end up producing an assembly will vary depending on overall sample quality, coverage, and relative abundance/degradation of each type of DNA.

Yes, any species can technically be sequenced and assembled with this method, but submitting samples for non-bacterial applications is at your own risk since we have not optimized the amount of data required for each specimen type and our assembly/annotation pipeline is targeted for bacteria. You may need to submit multiple aliquots of each sample in order to get enough genome coverage for these larger, more complex eukaryotic genomes (and note that you would need to combine data from all your aliquots prior to running your own assembly pipeline). If you are planning to submit a large number of samples for such an off-label application, contact us prior to submission to discuss options.

Custom Sequencing Service


If you are interested in sequencing a known mixture (e.g. barcode or variant libraries), please send us an email at support@plasmidsaurus.com to set up a custom project so that we can collect the amount of data you require.

Since our standard pipeline won't be able to generate a consensus from your mixture, we will send you the raw reads for you to analyze yourself; however we are able to use the newest ONT chemistry for these custom preps, which now delivers >99% accurate raw reads!

Probably! Email us at support@plasmidsaurus.com to discuss your project details.

In the vast majority of cases, we deliver custom sequencing results within 3-5 business days of receipt of your samples.

As per Oxford Nanopore’s specs for the chemistry and flowcells we currently use for custom project sequencing, the raw read accuracy is >99%. We do not provide a consensus or assembly for custom projects; we provide the raw reads and you need perform your own analyses, which will dictate your final data accuracy.

Custom projects require us tailoring our sequencing service to meet each customer's experimental needs. Please email us at support@plasmidsaurus.com to discuss and initiate your project.

The cost for each custom sequencing project is calculated as follows:

Total Data Required = Number of samples x Insert length x Number of species/barcodes/variants x Coverage required per variant
Project Cost = $500 base price for 1st Gb data + $50 for each extra Gb data + $50 for barcoding each sample

Custom projects start at $500 for up to 1 Gb of total raw data and add $50 for each additional 1 Gb. There will also be a $50 per-sample barcoding surcharge added for projects with more than 1 sample. We will provide you a price estimate (and custom quote if you need it) when you email us to discuss your project.

We provide only the raw reads (>99% accurate) for custom projects. You would need to perform analyses (demultiplexing barcodes, generating consensus, aligning variants, etc.) yourself.


I submitted plasmids and got amazing results! This changes the way I do my research!

Let your friends and social media followers know about us -- the more volume we get, the more we can lower our prices and turnaround time.
Have a suggestion for how to improve our service? Let us know at support@plasmidsaurus.com.

Just got my plasmid sequencing results back from @plasmidsaurus and the insert is perfecto. Even fixed some legacy mis-sequencing from looong ago. Only 3 SNPs after 10 years of use. I *highly* recommend their services and for everyone to sequence their *entire* construct. pic.twitter.com/7QOaANo5fa

— Sebastian S. Cocioba (@ATinyGreenCell) October 1, 2021

Don’t sleep on @plasmidsaurus for your whole plasmid sequencing. Price right. Data right. Turnaround time tight. And the auto annotation feature is a nice bonus. Plus these guys outta Eugene #oregonstrong #oregonlong…readsequencing https://t.co/vthGoROL0t

— Arpiar Saunders PhD (@your_arpy) October 1, 2021

Tired of Sanger sequence just a fraction of your plasmid? Having them failing because of not having a good annealing region? These guys are awesome and we use their ehoke-plasmid sequencing all the time. https://t.co/WAIgsvAX39

— Alex Bisson (@Archaeon_Alex) September 15, 2021

If you're looking for whole plasmid sequencing, definitely check out @SNPsaurus. Sent out samples Thursday evening and had (great) results back by midnight Friday. Very affordable and would highly recommend.

— Josh Currie (@_Josh_Currie) July 5, 2021

A surprising fraction of the plasmids we sequence have an unexpected assembly size. Here is a good example: https://t.co/Y9PyNMufuA

— SNPsaurus (@SNPsaurus) August 17, 2021