Bacterial Genome Sequencing

Sample type Category Size Concentration Minimum volume Price per sample Order now
Bacterial Genome Standard up to 7 Mb 50 ng/uL ≥20 uL $90 or 6 dinocoins Order
Big 7 - 12 Mb 50 ng/uL ≥20 uL $105 or 7 dinocoins Order
Dinocoins Pre-paid credits $15 Purchase

Description of Service

The bacterial genome sequencing service is intended for whole-genome sequencing and assembly of genomic DNA (gDNA) from a clonal population (single species) of bacteria. This service is performed using the latest long-read sequencing technology from Oxford Nanopore Technologies (ONT), and includes the following components:

  • Construct an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including minimal fragmentation of the genomic input DNA in a sequence independent-manner.
  • Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data is ~99.6% accurate and is delivered in .fastq format).
  • Produce a high-accuracy genome assembly (delivered in .fasta format) with Flye for assembly and medaka for polishing.
  • Produce a set of bacterial genome annotations with Bakta (delivered in various file formats).

You will receive the following data for bactereial genome sequencing:

  • .fasta file: We return a polished consensus sequence of the genome in .fasta format.
  • .fastq file: The raw .fastq sequencing reads.
  • Various other files: For genome annotations.

The target output of this service is a single high-accuracy, circular (when appropriate for the species), full-length bacterial chromosome contig of your bacterial species, with a set of bacterial genome annotations. However, our ability to deliver target output is directly dependent on the quantity, quality, and purity of the gDNA sent to us. We do not guarantee any specific output.

If we are not able to obtain:

  • 210 Mb of raw sequencing data for the “standard” service (i.e. 30x genome coverage of a single 7 Mb genome),
  • 360 Mb of raw sequencing data for the “big” service (i.e. 30x genome coverage of a single 12 Mb genome),
then our failure policy applies.

Preparing Your Bacterial gDNA Samples

This service requires 1 µg of high quality, high purity, high molecular weight (HMW), double-stranded gDNA, with recommended >50% of the DNA above 15 kb in length and recommended purity of 260/280 above 1.8 and 260/230 between 2.0-2.2. Our low sequencing prices and fast turnaround times do not include gDNA extraction services or gDNA quality control (QC) services, so it is your responsibility to verify that you are preparing suitable samples that meet these requirements prior to shipping them to us.

There are numerous approaches to bacterial culture, thus we do not provide specific recommendations. Please refer to published literature for protocols.

Any extraction method that yields high quality, high purity, high molecular weight (HMW), double-stranded gDNA that is free of nicks, gaps, breaks, and contaminants is suitable for this sequencing service.

Our in-house preferred method for extraction is the Zymo® Quick-DNA Miniprep Plus Kit (with a lysozyme pre-treatment for gram positive samples). Here are a few other extraction methods that we and others have had success with:

How to handle gDNA samples:

  • Avoid vortexing and fast or unnecessary pipetting; pipet with wide-bore tips only
  • Elute in elution buffer, not water
  • Do not expose to high temperatures (>37°C) for >1 hour, pH extremes ( <6 or>9), intercalating fluorescent dyes, or UV radiation
  • Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months
  • If using a speed-vac, avoid over-drying of gDNA; do not use heat in the speed-vac

  • Quantity: We require 1 µg of gDNA, either at a concentration of 50 ng/µL in 20 µL of elution buffer, or dried down with SpeedVac. Quantification assay MUST be performed with Qubit or equivalent fluorometric method (such as a plate reader).
    IMPORTANT: Do not use Nanodrop for quantification – spectrophotometric methods are NOT reliable for quantification!

    • HMW gDNA often requires extra homogenization effort (longer incubation time, increased incubation temperature, very extensive gentle mixing, etc.) to obtain accurate quantification. If separate DNA quantifications from the top and bottom of the sample are within 15% of each other, this is usually a good indication of adequate homogeneity.

    • If you have less than 1 µg of gDNA, we strongly recommend that you perform additional extractions to increase yield, but you may submit less than 1 µg at your own risk. Please email us to let us know about this before shipping your order, then prepare your gDNA at the same required concentration (50 ng/µL) but in a lower volume according to your total DNA yield.

  • Quality: We recommend gDNA samples with >50% of the total DNA mass contained within fragments above 15 kb in size. If the sample does not meet this metric, it would be necessary to repeat STEP 2.
    Size characterization may be performed with Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder (ideally with pulsed-field electrophoresis).

  • Purity: We recommend gDNA samples with 260/280 above 1.8 and 260/230 between 2.0-2.2. Purity assay may be performed with Nanodrop or other spectrophotometric methods. If the sample is contaminated and does not meet this metric, either re-extract the sample OR clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.
    IMPORTANT: Do not refer to the DNA concentration reported by Nanodrop as a substitute for the above required fluorometric quantification!

    • Must not contain RNA; we strongly recommend RNase treatment during extraction
    • Must not contain denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
    • Must not contain residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.)
    • Must not contain insoluble material or be colored or cloudy

Shipping Your Bacterial gDNA Samples

Please refer to our shipping instructions for details.