Eukaryotic Genome Sequencing


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Sample Type Category Size Concentration Minimum Volume Price Per Sample (USD)
Eukaryotic Genome 20 - 60 Mb* 50 ng/μL 20 μl $250
75 - 250 Mb* 50 ng/μL 40 μl $500
300 - 750 Mb* 50 ng/μL 60 μl $1000
1 - 3.3 Gb* 50 ng/μL 60 μl $1750
20 - 60 Mb* See Instructions Below $300
75 - 250 Mb* See Instructions Below $550
300 - 750 Mb* See Instructions Below $1050
1 - 3.3 Gb* See Instructions Below $1800

* These recommended genome sizes are based on an average target of ~30x genome coverage for de novo assembly. You can submit samples to a different size category if you need a different amount of data for your analysis application.

If your genome is larger than 3.3 Gb, please submit your sequencing request through Custom Sequencing instead - email us at support@plasmidsaurus.com to get started!

** A single PromethION R10.4.1 flow cell that is run for 72-96 hours is estimated (not guaranteed) to produce 50-100 Gb of basecalled data for high quality genomic samples.

*** This new eukaryotic extraction service uses a rapid, high-throughput protocol that is ONLY COMPATIBLE with the following approved list of input types that do not have a cell wall or abundant connective tissue. We can accept Shield-preserved samples from both BSL1 and BSL2 sources:

  • Cell culture from any animal species: Requires 5 x10^6 individual cells, pelleted out from growth media, washed once in 1x PBS, and resuspended in 200 µL of 1x Zymo DNA/RNA Shield.
  • Blood from most non-primate species: Requires 100 µL of blood (must contain nucleated cells), combined with 100 uL of 2x Zymo DNA/RNA shield, for a total sample volume of 200 µL. Please note we cannot accept blood samples from any primate species (e.g. human, monkey), and this service is not intended for clinical or diagnostic applications.

We currently do not accept any other input types (e.g. intact tissues/organs from any animal, plants, insects, fungi, primate blood) or preservation methods (e.g. flash freezing, ethanol, desiccation, RNAlater) for this extraction service. If you’d like to sequence these other types of samples, please perform extraction on your end and ship us only the purified gDNA for sequencing.

Description of Service

The Eukaryotic Genome Sequencing service is intended for long-read whole genome sequencing, assembly, and annotation of genomic DNA from any eukaryotic species with a genome size between 20 Mb and 3.3 Gb. Eukaryotic species that are ideally suited to this genome assembly service include filamentous fungi, protozoans, nematodes, fruit flies, zebrafish, mouse, and human.

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 eukaryotic genome assembly generated?)
  • Producing a set of eukaryotic genome annotations with Augustus (delivered in various file formats).

For pre-extracted gDNA we deliver genome sequencing results within:

  • 2-3 business days for the 1 Gb and 5 Gb Service Tiers
  • 4-6 business days for the 15 Gb and 50-100 Gb Service Tiers

When our eukaryotic DNA extraction option is selected, we deliver genome sequencing results within:

  • 4-6 business days for the 1 Gb and 5 Gb Service Tiers
  • 6-9 business days for the 15 Gb and 50-100 Gb Service Tiers

Higher data targets have longer sequencing and assembly times.

Applications for Service

This service is intended for the whole genome sequencing of double-stranded genomic DNA from eukaryotic genomes up to 3.3 Gb in size. All of our lab workflows, sequencing conditions, and data analyses for this service are optimized for the eukaryotic gDNA input.

Due to the rapid, high-throughput nature of this eukaryotic genome service, we are not able to make adjustments to the service parameters. If you’d like to request any of the following adjustments:

  • Sequencing more than 50-100 Gb of data (1 full flow cell) per sample
  • Sequencing a genome that is larger than 3.3 Gb
  • Sequencing an intermediate amount of data that does not have a service tier, such as 7 Gb
  • Splitting the data target per sample between multiple smaller samples
  • Using ligation-based library prep chemistry (instead of tagmentation)
  • Sequencing metagenomic (non-clonal) DNA from mixed species samples
  • Identifying variants between the samples or against a reference

… then please submit your request instead to our Custom Sequencing Service, where as always, we can collect the amount of data you request with the parameters you require!

Please note that other input types are not supported with this eukaryotic genome service, such as:

  • Other non-genomic, double-stranded DNA (e.g. plasmid or amplicon libraries)
  • RNA, single-stranded DNA, self-complementary AAV DNA, or other nucleic acids

Service Tiers (Categories): Choose Your Data Target

The Eukaryotic Genome Sequencing service offers four different Service Tiers (categories), which allow you to select the target amount of raw reads per sample, measured in Gigabases (Gb) of.fastq data. We do not specify an expected number of reads at these Service Tiers, as this will vary depending on the quantity, quality, and purity of the input gDNA.

The Service Tier recommendations below are based on a target of approximately 30x genome coverage, which is typically sufficient to generate a high-quality de novo genome assembly with annotations. Depending on your application, you can submit your sample to a different service tier than is recommended in order to obtain a different amount of coverage. For example:

  • If you plan to use the raw reads to perform structural variant analysis against a reference genome, you may need less genome coverage, e.g. 10-20x. Please note that assembly may fail at lower coverage levels, but you can ignore the assembly and just use the raw reads to perform your analysis.
  • If you plan to use the raw reads to generate a haplotype-phased de novo genome assembly, you may need more genome coverage, e.g. 60-100x. Please note that you would need to re-assemble the raw reads on your end to obtain haplotype phasing.
Service Tier (Category) Example Species Approx. Genome Size (haploid) Approx. Genome Coverage
1 Gb Plasmodium falciparum (protozoan) 23 Mb 43x
Fusarium oxysporum (fungus) 36 Mb 28x
Neurospora crassa (fungus) 43 Mb 23x
5 Gb Caenorhabditis elegans (nematode) 100 Mb 50x
Daphnia pulex (water flea) 125 Mb 40x
Drosophila melanogaster (fruit fly) 180 Mb 28x
15 Gb Anopheles gambiae (mosquito) 278 Mb 54x
Oryza sativa (rice) 430 Mb 35x
Mimulus guttatus (monkeyflower) 450 Mb 30x
50-100 Gb (1 flow cell) Danio rerio (zebrafish) 1.5 Gb 33-66x
Mus musculus (mouse) 2.7 Gb 18-36x
Homo sapiens (human) 3.3 Gb 15-30x

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.

  • .fastq.gz = a compressed file of all the raw ONT sequencing reads
  • .fasta = polished consensus sequence of the genome (may contain multiple contigs)
  • .gff = gene annotations for the polished genome
  • .html = A summary report compiling the assembly metrics, including completeness of the assembly based on Busco v5.7.1 and general species identification of the contigs. The metrics summarized in the report are also delivered as discrete files:
    • reads.png = histogram of all raw reads (indicating read length vs. Phred score), including coloration to distinguish reads that are retained for assembly vs. reads that are rejected
    • stats.tsv = metrics assessing the quality and size of the polished genome assembly and the raw reads that were used for assembly
    • busco-short-summary.txt = metrics assessing the completeness of the polished genome assembly
    • contigs.png = graph of the contig topology and their connections in the assembly
    • contigs.txt = metrics assessing the quantity and lengths of the contigs

The target output of this service is the specified amount of raw sequencing data, along with a high-accuracy contig or contigs for each chromosome and a set of eukaryotic genome annotations. However, our ability to deliver this target output is directly dependent on the quantity, quality, and purity of the gDNA or cells sent to us. We do not guarantee any specific output.

If we are not able to achieve these target outputs, then our Rerun & Failure Policy applies (see below).

Rerun & Failure Policy

The raw data targets are easily achievable when you send us high quality samples that meet our Sample Prep Instructions and Cell Prep Instructions. If your input has low quality, purity, and/or concentration, your samples may produce lower coverage and/or may fail to assemble; in cases of failure, you will still receive the raw reads we obtain, and potentially some of the other file types.

At the 1 Gb and 5 Gb Service Tiers, poorly performing samples are often rescued with our rerun procedure.

  • If a minimum of 75% of the data target (i.e 750 Mb and 3.75 Gb respectively) is not obtained on the initial sequencing attempt, samples at the 1 Gb and 5 Gb Service Tiers will be designated “fail” (regardless of assembly outcome) and will be automatically submitted for a free rerun to collect more data.
  • If the 75% minimum is still not met after two sequencing attempts, the sample will not be eligible for further repeats. If you’d like to collect more data, you would need to submit a new sample with higher quality on a new order.

At the 15 Gb and 50-100 Gb (full flow cell) Service Tiers, poorly performing samples are unlikely to be remedied even with our rerun procedure, so we save you money by stopping the run early.

  • We will stop the run early to preserve the lifespan of the flow cell. We will not attempt to perform a rerun on the sample.
  • Instead of the full $1750 flow cell price, you will be charged only $200 to cover our prep costs. If you’d like to try to collect more data, you can promptly request that we restart the run at your own risk (you would pay the full $1750 price regardless of outcome), OR you can submit a new sample with higher quality on a new order.

Preparing Eukaryotic gDNA Samples

This service requires high quality, high purity, double-stranded gDNA, at the specified volume & concentration listed in the table above, 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.

Any gDNA extraction method that yields high molecular weight (HMW), high quality, high purity, double-stranded gDNA that is free of nicks, gaps, breaks, contaminants, and inhibitors is suitable for this sequencing service. However, in general we recommend against using any phenol:chloroform extractions, as these typically have very poor sequencing performance.

Our in-house preferred method for extraction is the New England Biolabs Monarch® Spin gDNA Extraction Kit.

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

We require gDNA to be normalized in elution buffer to a concentration of 50 ng/µL, at the volume specified in the table above. Quantification must be performed with Qubit or an equivalent fluorometric method (such as a plate reader). Please DO NOT use Nanodrop for gDNA quantification, as it is not accurate enough.

  • HMW gDNA often requires extra homogenization effort (e.g. longer incubation time, increased incubation temperature, very extensive gentle mixing) 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 your extraction yields less than 1 µg of gDNA (i.e. less than 20µL @ 50 ng/µL), we strongly recommend that you perform additional extractions to increase yield, but you may submit a lower volume 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.

We recommend gDNA samples with 50% of the total gDNA mass contained within fragments above 15 kb in size. If the sample does not meet this metric, we strongly recommend repeating extraction to obtain higher quality gDNA. Size characterization may be performed with Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder (ideally using pulsed-field electrophoresis).

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, we strongly recommend either repeating extraction OR performing purification to remove the contaminants using AMPure XP beads.

  • Must not contain RNA; RNase treatment is required 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 Eukaryotic DNA Extraction Option

*** IMPORTANT - PLEASE READ ***

This new eukaryotic extraction service uses a rapid, high-throughput protocol that is ONLY COMPATIBLE with the following list of input types, which do not have a cell wall or abundant connective tissue:

  • Cell culture from any animal species
  • Blood from most non-primate species

We can accept Zymo 1X DNA/RNA Shield-preserved samples from both BSL1 and BSL2 sources of the above input types. We currently do not accept any other input types (e.g. intact tissues/organs from any animal, plants, insects, fungi, primate blood) or preservation methods (e.g. flash freezing, ethanol, desiccation, RNAlater) for this extraction service. If you’d like to sequence these other types of samples, please perform extraction on your end and ship us only the purified gDNA for sequencing.

NOTE: Our extraction service does not include growing your cells; we extract DNA directly from the preserved cells that you send us. It is important that a sufficient amount of cells have been preserved, otherwise the gDNA extraction will likely fail.

For cultured cells from any animal species without a cell well:

  • Count the cells to ensure you are sending 5 x 106 individual cells
  • Pellet the cells out from growth media
  • Wash the cell pellet once in 1 mL of 1x PBS
  • Resuspend the cell pellet in 200 μl of 1x Zymo DNA/RNA Shield to preserve cells
  • Transfer the 200 µL of preserved cells into a 200 mL strip tube

For blood from most animal species (excluding primate):

  • Pipet the blood to ensure you are sending 100 µL
  • Combine the blood with 100 µL of 2x Zymo DNA/RNA Shield (total sample volume = 200 µL) to preserve blood
  • Transfer the 200 µL of preserved blood into a 200 mL strip tube

Please note we cannot accept blood samples from any primate species (e.g. human, monkey), and this service is not intended for clinical or diagnostic applications.

Please place an order, selecting your desired Service Tier (category) and extraction options.

Please package and label your strip tubes containing preserved cells per our Shipping Instructions. Please send preserved cells at room temperature; dry ice and cold packs are not necessary.

Frequency Asked Questions & Interpreting Your Results

Please refer to our Results Interpretation Guide for details.