ZeroPrep Sequencing

Description of Service

Are you tired of performing MiniPreps to purify plasmids from your EndA- strains of E.coli that express high-copy plasmids up to 25kb in size? We can save you the hassle by performing whole plasmid sequencing directly from your bacterial colonies with our ZeroPrep service! This service produces an identical consensus sequence at the same quality as a purified MiniPrepped plasmid submitted to our Whole Plasmid Sequencing service.

sample .fastq file

Figure 1. Identical histograms and plasmid maps are generated from parallel MiniPreps (submitted to our Whole Plasmid Sequencing service) and ZeroPreps of the same bacterial culture.

Important notes:
  • ZeroPrep samples are not eligible for reruns.
  • ZeroPrep service is not compatible with:
    • Species other than E. coli
    • EndA+ strains of E. coli
    • Low copy plasmids
    • Plasmids larger than 25 kb

This service is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies , and includes the following components:

  • Constructing an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including linearization of the circular plasmid DNA within the cells in a sequence independent-manner.
  • Sequencing the library with a primer-free protocol using the most accurate R10.4.1 flow cells (raw data is >99% accurate and is delivered in .fastq.gz format).
  • Generating a high-accuracy circular consensus sequence from the raw reads.
  • Generating a set of feature annotations from the consensus sequence.

This service is intended for a clonal population of molecules. If there are multiple molecular species present, the pipeline will only return one consensus sequence for the single molecular species that produces the largest amount of total sequencing data. If you would like to sequence a known mixture (e.g. barcode or variant libraries), please purify the plasmids and submit instead to our Custom Sequencing Service. Please read more the topic of sequencing mixtures in our Results Interpretation Guide.

Data & File Types Delivered

When your results are ready, you will receive an email notification. Once you sign in to your account, you can download these results from your Dashboard. More detailed information about data & file types can be found in the Results Interpretation Guide.

  • Consensus sequence (.fasta file): Polished consensus sequence of the plasmid.
  • Consensus sequence (.gbk file): Polished consensus sequence of the plasmid, with a plasmid map and feature annotations.
  • Plasmid map (.html file): An interactive version of the plasmid map.
  • Read length histogram (.png file): Displays the read length distribution of the raw reads produced by your sample.
  • Virtual gel (.png file): Displays the raw read lengths from all samples in the order in a virtual gel format.
  • Chromatogram (.ab1 file): Displays the relative abundance of each nucleotide (A, T, G, C) for all raw reads that align to the consensus at each position of the sequence.
  • Coverage plot (.png file): Displays the relative sequencing coverage at each position of the consensus sequence.
  • Per-base data (.txt and .tsv files): Includes 3 sub-files for each sample:
    • SAMPLE.tsv: Indicates how well the raw reads agree with the consensus sequence at each position.
    • SAMPLE_multimer_analysis.txt: Indicates the % distribution of the various concatemer forms of the consensus sequence (monomer, dimer, trimer, etc.).
    • SAMPLE_summary.tsv: Indicates the length, average coverage, relative composition (by moles and mass), total reads, total bases, and %. E. coli genomic DNA contamination for the consensus sequence.
  • Raw read sequences (.fastq.gz file): Provides the sequences of individual raw reads that align to the consensus. Please note that these reads are NOT delivered in the default download, but can be downloaded separately by clicking the Download Raw FASTQ button at the top of the Order Information page. Also note that any raw reads that do not align to the consensus (e.g. host genomic DNA, lower abundance molecular species) are excluded.
Our ability to deliver these target outputs is directly dependent on the quantity and quality of the bacterial cells sent to us and the plasmid they contain, so we do not guarantee results.

Preparing Your Cells for ZeroPrep

We accept EndA- E. coli cells in any of the following 3 configurations:

A. 1 large colony from agar plate, picked and resuspended in 10 uL of water, shipped in 200 μl strip tubes.

  • The sample should be obviously cloudy, the cloudier the better. These can be processed on the day of receipt and yield overnight results.

B. 100 μl of liquid culture, shipped in 200 μl strip tubes.

  • We will grow overnight at 37°C if the culture isn't dense.

C. Circled and numbered colonies, shipped on agar plate.

  • Provide us with E. coli colonies spotted on an agar plate. It's easiest to streak a 5 mm spot from each colony onto the plate, and then circle and number the colonies you want sequenced. We will incubate the plate overnight at 37°C before processing if the colonies are too small or not visible.

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.