plasmidsaurus

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
	Standard with extraction	up to 7 Mb	Send a bacterial pellet in DNA/RNA Shield		$105 or 7 dinocoins	Order
	Big with extraction	7 - 12 Mb	Send a bacterial pellet in DNA/RNA Shield		$120 or 8 dinocoins	Order
Dinocoins	Pre-paid credits				$15	Purchase

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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% 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).

In the vast majority of cases, 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 1 week of receipt of your samples.

You will receive the following data for bacterial 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 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

Preparing Cell Pellets for the Bacterial DNA Extraction Option

All samples must be sent as cell pellets that have been resuspended in DNA/RNA Shield. We accept both BSL1 and BSL2 strains.

We do not grow your samples, we extract the DNA directly from the material you send us. It is important that enough cells have been collected, otherwise the DNA extraction will likely fail.

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. Do not send cells from older cultures that are in death phase.

We require the equivalent of 8-12 OD600nm or 4-6 x 10^9 cells (e.g. 8-12 mL culture at 1.0 OD600nm). A compact cell pellet with all the supernatant removed (e.g. E. coli pellets) should be approximately 15 mg. A wet cell pellet where all the supernatant cannot be removed without disturbing the pellet (e.g. Streptococcus sp. pellets) should be approximately 30-50 mg. 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.

The cells should be pelleted by centrifugation, washed with 1 mL PBS and pelleted again.

Place an order on our website and receive a 3-character code. When filling out your sample names please be sure to indicate if your strain is gram positive or gram negative by adding either a plus (+) or a minus (-) at the end of your sample name. Failure to do so may result in failure of the DNA extraction.

Resuspend the final pellet in 0.5 mL of 1X DNA/RNA Shield in a 2 mL screw cap tube. Please label each tube with the order label and sample number and either a plus sign (+) or a minus sign (-) to indicate if the strain is gram positive or negative. For example, "X2X1-", "X2X2-", etc,

To prevent damage and leakage of the tubes during shipment, we ask that you put the tubes in a rigid container such as a falcon tube or tube box before shipping. For orders of more than 10 samples we ask that you 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 samples at room temperature.

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

Shipping Your Bacterial gDNA Samples