Long-Read Amplicon Sequencing and Analysis

Service Description

Our long-read amplicon sequencing and analysis service provides comparative analysis of your amplicons to a reference sequence you provide. Using a rapid, cost-effective, long-read sequencing technology, we are able to detail the entirety of the nucleotide sequence including any variants that were observed. The methodology allows for amplicon sequencing of lengths >1kb without the need for multiple PCRs spanning the target of interest as required with Sanger or short-read sequencing. This approach also allows variants to be phased.* Deliverables include a thorough HTML report, a session file in XML format for use in IGV, FASTQ files of sequence reads, and a BAM file containing read alignments.

*  Phased variant analysis is not included in the basic report and is contingent on sufficient numbers of contiguous reads and variants. Phasing analysis may be requested of the Bioinformatics Resources Core. Please see our Guidelines and FAQs for how you can have the greatest success with amplicon sequencing.

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 amplicons are submitted in a 96-well plate.

Important Notes and FAQs

What do I get back from UWBC? We will provide a thorough HTML report, a session file in XML format for use in IGV, fastq files of passed reads, and a BAM file detailing variants. A zipped folder containing example data can be found here (if the file doesn’t download, copy/paste the URL into a new browser window).

How do I best view the data? It is best to use Integrative Genomics Viewer (IGV). You can open a new session using the provided XML file. Tutorial videos for IGV can be found here.

Can you analyze SNPs or indels in my amplicons? Yes! For greatest success, your amplicon should be >500bp and your primers should be designed at least 200 bp outside your target(s) of interest. The reference sequence you provide should begin with your forward primer and ends with your reverse primer. Your reference sequence should be the WT sequence to which your SNPs or indels will be compared.

Does UWBC perform QC of my amplified DNA? No upfront QC of the input DNA or QC of the final library is performed. Ensure the following before you submit.

  • Optimize your reaction to avoid primer dimer, spurious products, over-amplification/heteroduplex formation, or weak enrichment.
  • Check for effective target amplification. Verify the presence of the intended product and absence of primer dimers or spurious products by visualization on an agarose gel. Any and all dsDNA products submitted to us have the potential of being sequenced.
  • If you are having trouble with your initial amplification, contact the DNA Sequencing facility before you submit.

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

When can I expect results? If your amplicon 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.

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 amplicon sequencing. High quality inputs that meet specifications are very likely to get more than sufficient coverage. We don’t guarantee variant detection, phasing of variants, minimum read count, or minimum/even amplicon coverage. If you aren’t seeing what you expect in your data, there may be issues other than quality and quantity to consider such as complexity of the amplicon (specifically, but not limited to, homopolymeric regions). This may be addressed with other methods – we’re here to help!

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

Why do I see indels and frameshifts in my amplicon sequence? 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 reads, these regions are still prone to single nucleotide indels.

Why should I choose Long Amplicon Sequencing and Analysis over other methods? This service is great for cost efficient screening without having to perform primer walking, cloning, and other laborious techniques. Furthermore, you don’t need to compromise on read length or settle for sequencing only portions of your amplicon. Provided you have successful amplification, the entire amplicon can be sequenced and analyzed for variants!

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 what I expected in the report I received? Reasons you may not see expected results vary but are likely due to the provided product not being what you intended to amplify (spurious amplification or pseudogene), insufficient product provided, or incorrectly provided primer and/or reference sequences. Below are some examples of these scenarios.

“Perfect” amplicon

Insufficient/poor amplification

Multiple amplicons (doesn’t imply failure, but may fail due to desired/referenced target being poorly amplified)

No amplification

Incorrectly specified primers or reference sequences

 

 

 

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.