The bacterial genome sequencing service is intended for whole-genome sequencing and assembly of
(gDNA) from a clonal population (single species) of bacteria. This service is performed using
long-read sequencing technology from Oxford Nanopore Technologies (ONT), and
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
Sequence the library primer-free using the most accurate R10.4.1 flow cells (raw data
accurate and is delivered in .fastq format).
Produce a high-accuracy genome assembly (delivered in .fasta format) with Flye for assembly and
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
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,
and purity of
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
360 Mb of raw sequencing data for the “big” service (i.e. 30x genome coverage of a single
12 Mb genome),
This service requires 1 µg of high quality, high purity, high molecular weight (HMW), double-stranded
of the DNA above 15 kb in length and recommended purity of 260/280 above 1.8 and
between 2.0-2.2. Our low
sequencing prices and fast turnaround times do not include gDNA extraction services or gDNA quality control
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.
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,
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
had success with:
Avoid vortexing and fast or unnecessary pipetting; pipet with wide-bore
Elute in elution buffer, not water
Do not expose to high temperatures (>37°C) for >1 hour, pH
<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
quantification – spectrophotometric methods are NOT reliable for quantification!
HMW gDNA often requires extra homogenization effort (longer incubation time,
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
same required concentration (50 ng/µL) but in a lower volume according to
your total DNA
Quality: We recommend gDNA samples with >50% of the total DNA mass
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,
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
2.0-2.2. Purity assay may be performed with Nanodrop or other spectrophotometric methods. If
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
by Nanodrop as a substitute for the above required fluorometric quantification!
Must not contain RNA; we strongly recommend RNase treatment during
Must not contain denaturants (guanidinium salts, phenol, etc.) or
detergents (SDS, Triton-X100, etc.)
Must not contain residual contaminants from the organism/tissue (heme,
Must not contain insoluble material or be colored or cloudy