Plasmid Sequencing and Assembly

Service Description

Our plasmid sequencing and assembly service aims to provide you with a complete view of your plasmid and allows for characterization of the target gene and verification of the plasmid backbone. Confirming orientation and number of tandem repeats of the insert and presence of promoters, cut sites, and origin of replication are simultaneously possible. Not only will you receive a FASTA file containing the consensus sequence of your plasmid and an annotated map of your plasmid, you will receive greater assurance that your construct can be used in downstream applications.*

*  There is no guarantee of plasmid assembly. Please see our Guidelines and FAQs for how you can have the greatest success with assembly of your plasmid. Where greater nucleotide resolution or independent confirmation is necessary, we offer other services that can help resolve assembly of difficult plasmids!

Access our Interactive Submission Guidelines and get the exact guidance you need to prepare, label, and deliver your samples.

 

If you can’t find the information you’re looking for, please contact us at nextgen-seq@biotech.wisc.edu

Service Pricing

* Flat rate pricing is ~$13/sample (UW System) with 96 samples. Flat rate pricing will be charged when plasmids are submitted in a 96-well plate.

Important Notes and FAQs

Does UWBC perform QC of my plasmid DNA? No upfront QC of the input DNA or QC of the final library is performed. We highly recommend that you perform fluorometric quantification (e.g. Qubit) of your plasmid before submitting to ensure you meet the specifications listed in our guidelines.

Do you hold onto my plasmid and can I get it back? Once the data is delivered, we dispose of the plasmid. We suggest you retain sufficient plasmid for your needs as we will not send it back to you.

When can I expect results? If your plasmid is submitted and delivered to us by 8am on Tuesday, the data will be delivered by Thursday of that week. The exception may be weeks with intervening holidays where the facility is closed. If the sample is later than 8am on Tuesday, it will be processed the following week. Please note that shipments received at our loading dock on Tuesday will not reach us in time – please plan shipments accordingly.

What do I get back from UWBC? For each successfully sequenced and assembled plasmid, we will provide a pLannotate map and FASTA file. Soon, we will be providing a custom report with additional details! More information on pLannotate can be found here.

How do I access my data? Our preferred method to distribute data is through Globus (external clients) and ResearchDrive (internal clients). More information can be found here. You will receive an email when your data is available and it will contain instructions on how to access it.

How long do you store my data? We store your data for 60 days. After 60 days, it is deleted from our servers. Please download your data as soon as you receive the data delivery notification!

Do you guarantee success or read counts? Following our guidelines gives the highest chance of successful plasmid assembly. High quality plasmids that meet specifications are very likely to get more than sufficient coverage. We don’t guarantee successful assembly, circularization, or annotation of the plasmid or minimum read count or coverage. If your plasmid failed to assemble, there may be issues other than quality and quantity to consider such as complexity of the insert. This may be addressed with other methods – we’re here to help!

Do you still charge if no data is generated? Each plasmid will be charged, even if no data is generated. Refer to our guidelines for the best chance of a successfully assembled plasmid.

Why do I see indels and frameshifts in my assembly? These are usually associated with homopolymeric nucleotides. The length and frequency of homopolymers increases the occurrence of indels. Even though error correction is performed on the assembled reads, these regions are still prone to single nucleotide indels.

Why should I choose Plasmid Sequencing and Assembly over other methods? This service is great for cost efficient screening without having to design primers. The entire plasmid can be sequenced, assembled, and annotated, providing greater confidence in use of your plasmid in downstream applications!

What type of basecaller is used? Guppy basecaller with the Super accurate (SUP) model is used, which is the most accurate model available for Oxford Nanopore data.

Why do I not see a single, “perfect” peak in the summary report (.html) for read length distribution? Reasons for seeing multiple peaks include overall low sample quality/purity and specifically, plasmid degradation and/or nuclear DNA contamination. Below are some examples of these read length histograms.

Perfect” Plasmid

Concatemers

Degraded Plasmid

Multiple Templates

 

 

 

Unless otherwise specified, all data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Sequencing Facility are intended for research purposes only. They are not intended nor certified for diagnostic or clinical use. Clinical services are provided through our collaboration with the UW Collaborative Genomics Core.