FREQUENTLY ASKED QUESTIONS

We are always excited to hear customer requests for new samples types and explore those in custom projects. In the near future we hope to offer more sample types. Stay tuned!

We use Nanopore Technology (ONT) long-read sequencig technology to sequence our customer samples. We first construct a sequencing library using ONT Kit v14 library preparation chemistry. During this step, double-stranded, circular plasmid or linear amplicon DNA is linearized and attached to barcodes in a sequence-independent manner. The library is then sequenced, without primers, on R10.4.1 flow cells with raw data accuracy >99%. As a last step the raw reads are run through our bioinformatics pipeline to create a high accuracy assembly that is fully annotated. All samples are sequenced without any amplification steps or primers. Hence, please avoid sending primers along or inside your samples.

It is imperative that fluorometric methods such as Qubit are used to accurately quantify DNA samples. Absorbance-based methods such as Nanodrop are NOT adequate and will likely result in sequencing failure due to inadequate DNA quantities

No, Nanodrop is NOT SUFFICIENT for DNA quantification because of its limited accuracy. The discrepancy in Nanodrop measurements can significantly vary depending on the unique composition of your sample, making it impossible to establish a standard method for adjusting Nanodrop values. Submitting samples with excessively high or low concentrations based on Nanodrop values might negatively impact the library preparation and/or sequencing reactions, potentially leading to sequencing failure.

You can either send your samples to us directly or use one of our dropboxes at your institution. We currently sequence every Tuesday and Friday. Please closely follow our Submission Guide for details on sample preparation and shipping.

You can check the received status of your order under View orders. In case your order does not show as received, your samples have not yet reached us, or we might still be unboxing the delivered parcels. Keep monitoring your order for any updates.

Please reach out to our support team for the latest list of sample dropboxes or to request a new dropbox at your location. If you are sending your samples directly to us, they need to arrive at our laboratories before Tuesday 13:00 or Friday 9:00to be included in the days sequencing run.

You can either pay with Unveil Bio credits or by credit card via our partner Stripe. Please follow the payment instructions after creating an order. If you require alternative payment methods please reach out to support@unveil.bio.

Unveil Bio credits can be used to pay for your sequencing orders. One credit equals one small plasmid sample. You can buy credits in bulk to avoid having to enter your payment details for every order. Volume discounts are available. Please reach out to support@unveil.bio if you would like to purchase credits.

Please check our Submission Guide for information on how to submit sequencing orders.

No, we accept any number of sample(s).

Yes, absolutely!

Yes, absolutely! Please check your local requirements for shipping DNA samples.

With Unveil Bio's whole plasmid sequencing you get many benefits over traditional short read sequencing of only your region of interest:

• No primers needed
• Low input DNA amount
• Detect E. coli genomic DNA contamination
• Detect multiple plasmid species in the same sample
• Ensure scientific accuracy and confidence
• E. coli and other host organisms might employ various strategies to avoid expressing a burdensome or toxic gene. These strategies can involve altering the plasmid in ways that targeted Sanger sequencing cannot detect.
• Whole plasmid sequencing is more convenient than multiple Sanger runs, custom sequencing primer synthesis, or primer walking.
• Long-read sequencing is perfect for deciphering repetitive regions that can be challenging for Sanger sequencing to resolve.
• Sanger sequencing might not reveal if your plasmid is a dimer or if there are multiple plasmids within your strain, but we come across such cases regularly.
• Whole plasmid sequencing is similar in price and time compared to other methods.

We aim to have a turnaround time of less than 3 days. For Tuesday sequencing, results are ready by Friday. For Friday sequencing, results are ready by Monday.

Oxford Nanopore Technology has come a long way and now enables high accuracy comparable to Sanger sequencing:

• > 99% raw read accuracy
• > 99.999% / Q50 consensus accuracy

Nanopore's primary error types include deletions within homopolymer sequences and mistakes at the central position of Dcm methylation sites, such as CCTGG and CCAGG. Improvements to Oxford Nanopore's sequencing chemistry and basecalling software in future updates should help address these limitations.

Many of our customers employ E. coli as their preferred propagation host. Accordingly, we align all reads to the E. coli K-12 genome and then provide a percentage of the reads that successfully mapped. A clean DNA preparation (or a preparation from a host other than E. coli) will often yield a value below 1%. However, when cloning plasmids with E. coli pathways, this percentage may be misleadingly high because we do not differentiate between chromosomal DNA and the DNA from the cloned plasmid.

• .fasta file: A polished consensus sequence of the plasmid in fasta format.
• .gbk GenBank file: An annotated pLannotate plasmid map in GenBank format. Positions below our QC thresholds are marked as annotation and indicate low confidence in the accuracy of the indicated base; LOW-QUALITY, LOW_COVERAGE, POSSIBLE-VARIANT.
• quality.pdf file: A quality report in pdf format that shows the number of times a base has been sequenced in grey bars (Coverage) and a quality score for each base position as a cyan coloured line (FASTQ PHRED Quality). The latter is an internal quality metric where high values indicate a high confidence in the accuracy of the identified base. In addition, the quality report shows nucleotide variants that were found at each sequence position and their abundance is indicated by the height of the coloured bar. E.g.: If sequence position 256 has a grey bar for base T and 30% of the bar is coloured green then 30% of the sequence reads had an A at this position and 70% had a T. Bases that represent >60% of all base variants found are colored in grey.
• quality.tsv: The same information as in the quality.pdf file but in tsv format which can be opened in Excel or similar
• read lenght plot: A histogram showing the number of sequencing reads and their length. Bar colors indicate whether the reads mapped to the assembly, E. coli chromosome, or neither. The dotted vertical line indicates the assembly size. Ideally, the majority of sequencing reads should have a legnth equal to the assemly length which indicates that the entire plasmid was sequences in single continuous reads.
• .html pLannotate map: An annotated plasmid map, generated using the pLannotate tool from the Barrick Lab.