Bacterial Genome Sequencing

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.

When you send pre-extracted gDNA, we deliver bacterial genome sequencing results within 1-2 business days of receipt of your samples. When our genomic DNA extraction option is selected, we deliver bacterial genome sequencing results within 3-5 business days of receipt of your samples. When the Hybrid sequencing option is selected, we deliver hybrid results with 6-8 business days (or 8-10 business days if you also include the extraction option).

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

  • Constructing an amplification-free long-read sequencing library using the newest v14 library prep chemistry, including minimal fragmentation of the gDNA 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 delivered in .fastq format).
  • Producing a high-quality genome assembly (How is the bacterial genome assembly generated?)
  • Producing a set of bacterial genome annotations with Bakta (delivered in various file formats).

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.

  • FASTA files:
    • .fna (contig nucleotide sequences) = polished consensus sequence of the genome
    • .faa (protein amino acid sequences)
    • .ffn (gene nucleotide sequences)
  • GenBank files:
    • .gbff (annotated contig sequences) = polished and annotated consensus sequence of the genome
  • FASTQ file:
    • .fastq.gz = a compressed file of all the raw ONT sequencing reads
  • Report:
    • .html = An analytical report of the key metrics for the assembly (including completeness of the assembly based on CheckM and general species identification of the contigs)
  • Various other Bakta annotation files

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

Successful sequencing is defined by achieving at least one of the following deliverables:

  • A high-quality genome assembly
  • Target amount of ONT data
    • 210 Mb of ONT sequencing data for the "standard" service (i.e. 30x genome coverage of a single 7 Mb genome)
    • 360 Mb of ONT sequencing data for the "big" service (i.e. 30x genome coverage of a single 12 Mb genome)

If we are not able to achieve at least one of those deliverables, then our repeat policy applies. Even when a high-quality assembly cannot be generated, we still provide the raw data and the report, and you may also still receive some of the other file types.

We are now offering a Hybrid ONT + Illumina bacterial genome sequencing service that polishes your long-read ONT assembly with Illumina reads. We use the Polypolish v0.6 tool to improve the consensus quality at those tricky homopolymer runs and methylation motifs. The options for this service (standard and big genomes, with and without extraction) are listed in the order table on the Dashboard.

In addition to the ONT-only target deliverables listed above (ONT-only assembly and files), this hybrid service adds on these additional target deliverables:

  • Target amount of raw Illumina data (you will receive the .fastq reads):
    • 210 Mb of Illumina data for the "standard" service
    • 360 Mb of Illumina data for the "big" service
  • Polished .fasta assembly reference file generated with Polypolish v0.6.0, with the ONT .fna file and Illumina .fastq files as input

The input DNA requirements for this hybrid service are identical to the ONT-only requirements, as detailed below. You do not need to send extra DNA!

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 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 methods for extraction are the Zymo® Quick-DNA Miniprep Plus Kit (with a lysozyme pre-treatment for gram positive samples) and Zymo® Quick-DNA Fungal/Bacteria Miniprep Kit. 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, at a concentration of 50 ng/µL in 20 µL of elution buffer. Quantification should be performed with Qubit or an equivalent fluorometric method (such as a plate reader). We strongly discourage using Nanodrop for gDNA 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, we strongly recommend repeating 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.

    • 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

Preparing Cell Pellets for the Bacterial DNA Extraction Option

All samples must be sent as cell pellets that have been resuspended in Zymo 1X DNA/RNA Shield preservative. Please do not use any other type of preservation media, such as RNAlater, as they do not preserve the DNA as effectively and do not fully inactivate the host cells, which is required for safe handling in our lab. We accept cell pellets from both BSL1 and BSL2 strains.

We strongly recommend growing a fresh clonal culture of your bacteria in liquid broth and harvesting the cells when they are in exponential growth or early stationary phase. Please do not send cells from older cultures that are in the death phase.

NOTE: Our extraction service does not include growing your samples; we extract DNA directly from the material you send us. It is important that enough cells have been collected, otherwise the DNA extraction will likely fail.

Due to customs regulations, the bacterial extraction service is currently only available to US customers.

1. Pellet the cells by centrifugation and remove as much supernatant as possible.

We require the equivalent of 8-12 OD600 or 4-6 x 109 cells (e.g. 8-12 mL culture at 1.0 OD600).

  • A compact cell pellet with all the supernatant removed (e.g. E. coli pellets) should weigh approximately 15 mg.
  • A wet cell pellet where supernatant cannot be all removed without disturbing the pellet (e.g. Streptococcus sp. pellets) should be approximately 30-50 mg.
  • Please DO NOT send more than 50 mg of material or there will be too many cells to be protected from degradation by the DNA/RNA Shield.
2. Resuspend (wash) the pellet in 1 mL PBS, then pellet again and remove supernatant.
3. Resuspend the final pellet in 0.5 mL of Zymo 1X DNA/RNA Shield in a 2 mL screw cap tube.

Please place an order, either for Standard with extraction (up to 7 Mb) or Big with extraction (7-12 Mb).

When filling out your sample names, please add either a plus (+) or a minus (-) at the end of each sample name to indicate whether each strain is gram positive or gram negative. Not doing so will cause a processing delay for your order, as we need this information directly from you.

Please label each 2 mL screw cap tube with the order code, sample number, and the plus (+) or a minus (-).
For example, "X2X1-" would indicate order code X2X, sample 1, gram negative.

To prevent damage and leakage of the tubes during shipment, please place the screw cap tubes in a rigid container such as a falcon tube or tube box before shipping. For orders of more than 10 samples, please put the tubes in a tube box and load the samples row by row in numerical order as this greatly reduces the handling time when receiving your samples.

Please send preserved cell pellets at room temperature. Dry ice and cold packs are not necessary.

Interpreting Your Results

Please refer to our Results Interpretation Guide for details.