We use Nanopore Technology (ONT) long-read sequencing technology to sequence our customer samples. We first construct a sequencing library using ONT Kit v14 library preparation chemistry. During this step, circular plasmid DNA is linearized 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.

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.

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

• .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.