What service do you provide, what can we expect and what is the turnaround time?

We provide an Oxford Nanopore Whole Plasmid Sequencing Service. We sequence your plasmid without primers and send you your data with the assembled plasmid, annotated plasmid maps and some quality graphs for you to troubleshoot with if there are any issues. We currently run evening sequencing runs Monday-Friday with overnight results (before midday the following day). There may be some delays if we have a flow cell failure, but the hope is that this would only delay the data by a few hours.

What files do you provide as part of the results?

  • Assembled plasmid file (.final.fasta)
  • Annotated genbank file and html plot (.final_pLann.html and .final_pLann.gbk)
  • Raw basecalled sequencing data (_raw.fastq.gz)
  • Length and quality plots for your raw reads (_length.png and _qual.png)
  • Statistics file used to generate the length and quality plots (.stats)
  • As a final check we align the raw basecalled reads against your assembled plasmid file and provide a file that shows the proportion of bases that correspond to the assembled plasmid fasta by position (_alignment.csv). We place potentially problematic positions at the top of this file (where the proportion corresponds to less than 80% of the total number of reads covering that position. We also provide a visual representation of this data (_bam_alignment.png) and a format compatible with bioinformatics software to quickly visualise the quality (.fastq).

Why did my sample not yield the expect plasmid size?

Sometimes we find that there are no plasmid reads that correspond with the size that you expect. This can be because your cloning has not worked and you have an empty backbone, however, we also commonly see plasmid concatamers and mixed samples. The best way to troubleshoot this is to look at the _length.png file that we provide. If you have a single plasmid you expect to see the below graph with a single peak and minimal genomic DNA contamination.

Image of our control showing a single histogram peak at the approximate length of the expected plasmid

Why didn't I receive an assembled plasmid map?

The most common failure modes for nanopore whole plasmid sequencing are (1) genomic DNA contamination and (2) too low a concentration. We often find that there is substantial genomic DNA contamination and this essentially leads to low effective plasmid concentrations. Although other providers recommend sending Qubit quantified DNA we find that this can be quite the ask for most groups. We ask that you run an electrophoresis gel when you first send us samples to check the quality of your plasmid preparations and ensure the spectrophotometrically measured DNA concentrations make sense given what you see on your gel. Genomic DNA contamination will look like the graph below in the _length.png plot that we send you.

Image of our a sample showing short reads that are not close to the length of the expected plasmid size

Why do I seem to get the same errors in my assembled plasmid maps?

Nanopore has two "failure modes" when it comes to basecalling. The first is homopolymers regions (polyA, G, T and C) where it often is not able to tell how long the stretch is as the current doesn't change while the strand moves through the pore. The second is methylated regions where although there are basecalling models to call base modifications