Dear Customer,

Thank you for your interest in NRGene™ DeNovoMAGIC™ or DeNovoMAX services.

We look forward to establishing our collaboration and provide you with a fast, accurate, and cost-effective genome assembly. Our genome assemblies are well known in the scientific community as well as in the commercial market for their high standards and quality.

Optimal genome assembly rely on high quality gDNA extraction which is suitable for library preparation followed by sequencing and assembly. In order to make sure you will get the most satisfying results, kindly adhere to the following standard instructions and specifications.

Please feel free to address us with any inquiry with the bellow email address,

Thank you for the collaboration,

NRGene professional services team.

gas_support@nrgene.com

DNA extraction specification

High quality gDNA Extraction

Isolation of intact, double stranded, highly concentrated and uncontaminated genomic DNA is a prerequisite for successful and reliable DeNovoMAGIC or DeNovoMAX project.

Below you will find NRGene™ specifications for gDNA extraction:

Zygosity

DNA should be extracted from a single individual (highly recommended) or alternatively from serval clones produced by vegetative reproduction of a single individual. *DeNovoMAX is limited to homozygous species.

Homozygous species – in case an extraction from a single individual (or vegetative clones) is not feasible, extraction can be made by an equimolar mix (equal amounts of tissues) of at least 10 individuals of the same genotype (this will reduce contamination risk and problems in downstream processes).

Heterozygous species - DNA should be extracted from a single individual (highly recommended) or alternatively from serval clones produced by vegetative reproduction.

Extraction Specifications and Quality

Please note each sample should be prepared from the same DNA batch this is essential for downstream analysis procedures

Parameters	Thresholds	Recommended analysis method
DNA concentration	> 30 ng/µl	fluorometric methods as Qubit™ (Nanodrop is not suitable)
DNA total mass	For short reads libraries, DeNovoMAGIC: Genome size <3Gb, 30 µg Genome size >3Gb, 40-100 µg (based on genome size) For short reads libraries, DeNovoMAX: Genome size <3Gb, 10 µg Genome size >3Gb, 15-35 µg (based on genome size) For synthetic long reads (10X) library: Genome size <3Gb, 2 µg Genome size >3Gb, 2-5 µg (based on genome size) For long reads library (optional): Genome size <3Gb, 6 µg Genome size >3Gb, 6 µg for every 3Gb increment in genome size
HMW-DNA	For short reads libraries: 70% of DNA is above 30kb For synthetic long reads (10X) library: 70% of DNA is above 80kb For long reads library (optional): 70% of DNA is above 50kb	Agarose gel (0.8%, ladder above 48.5Kb); Fragment Analyzer™; TapeStation™; PFGE
Purity - RNA	without any traces of low molecular weight bands in the gel	Agarose gel (0.8%, ladder bellow 48.5Kb); Fragment Analyzer™; TapeStation™; PFGE
Purity - protein & organic solvent	260/280 ratio between 1.8- 2.2 260/230 ratio between 1.9- 2.2	Nanodrop™ (Thermo-scientific) or equivalent

Organism	Recommended protocol	Comments
Plant	CTAB based method	Use very young etiolated tissue, alternatively use leaves that have just finished budding
Animal	Trysol based protocol / DNA extraction kit for animal

NRGene recommendations for High Molecular Weight DNA extractions methods:

Please note: Should you encounter difficulties in gDNA extraction, or adhering the standards mentioned above, kindly contact NRGene support team.

Our Genomic Analysis Services may assist you with their own expertise or may refer you to a 3rd party service provider that might perform the DNA extraction for you.

e-mail: GAS_support@NRGene.com

QC by NRGene

NRGene will perform a preliminary assessment of the DNA samples prior to shipment to the sequencing providers. Please provide a filled and signed Certificate of Analysis (“COA”) shown in Annex A to NRGene’s personnel for review (GAS_support@NRGene.com). The COA should be signed and filled with the actual measured values and attached with a high-resolution picture of an agarose gel or other QC results available.

Samples Shipment Instruction

DNA samples should be stored in a controlled temperature to avoid degradation and/or breakage of the high molecular weight DNA molecules, shipping should also be handled carefully to avoid spillage or shear stress.

Storage

Dissolve the DNA in TE buffer (10mM tris, with a maximum of 0.1mM EDTA). The samples may be stored at 4°C for up to 2 weeks, or at -20°C (on dry Ice) for up to 6 months.

Important notice: Avoid freeze-thaw cycles as much as possible, it might cause a severe accelerated degradation and damage to the gDNA

Labelling

All samples should include the following information:

Project number

Organism Name

Variety name

Sample number (or name)

For example, label Project_85_Wheat_A.tauschii_3 will be for: project number (85), organism name (Wheat) variety (A.tauschii) and sample number (3).

*Label format should exclude spaces – please use only underscores “_”

Some samples may require additional documentation as instructed by NRGene personnel such as a reservation number(s) or declarations of non-hazardous materials.

Shipping

Samples that are stored at a 4°C temperature should be shipped on ice using a temperature-controlled package (4-8°C).

Frozen Samples should be shipped on dry ice, with sufficient amount to maintain the samples frozen until their arrival.

Important notices:

Avoid freeze-thaw cycles as much as possible, it might cause a severe accelerated degradation and breakage damage to the gDNA

Shipment is advised to take place at Monday-Wednesday to avoid long weekend storages that might cause frozen samples to defreeze and temperature abuse during storage.

Shipment is advised to be with a well-known currier company (e.g. FedEx). Shipments via small companies might cause delays and get held in customs for longer than usual.

Shipments should be accompanied with the proper documentation- consult with the carrier regarding customs requirements.

Tracking

Samples should be shipped with a well-known (A class) carrier which will provide tracing information and coordinate their reception.

Annex A- DeNovoMAGIC™ & DeNovoMAX

Certificate of Analysis (COA)

Kindly fill in the table below with values measured by the specified method and/or equipment and attach pictures of Agarose Gel/fragment analyzer/Tape Station for DNA purity/DNA molecular weight tests.

Project #	DeNovoMAGIC or DeNovoMAX	Organism name	Variety name/number	Haploid genome size [Gbp] (±15%)	Zygosity	Ploidy
Extracted from a single plant, grown in dark for at least 2 days? ☐Yes ☐No If not, please explain: ____________________

Parameters	Expected Measurements	Actual Measurements	Analysis method
DNA concentration	> 30 ng/µl	fluorometric methods as Qubit™ (Nanodrop is not suitable)
DNA total mass	For short reads libraries: For synthetic long reads (10X) library: For long reads library (optional):
HMW-DNA	70% of DNA is above 30kb (short reads) 70% of DNA is above 80kb (10X) 70% of DNA is above 50kb (long reads, optional)	Agarose gel (0.8%, ladder above 48.5Kb); Fragment Analyzer™; TapeStation™; PFGE
Purity - RNA	without any traces of low molecular weight DNA/RNA	Agarose gel (0.8%, ladder bellow 48.5Kb); Fragment Analyzer™; TapeStation™; PFGE
Purity - protein & organic solvent	260/280 ratio between 1.8- 2.2 260/230 ratio between 1.9- 2.2	Nanodrop

Notes:

Project number is the quote number provided to you by NRGene sales-manager (e.g. 123)

Agarose gel pictures for 10X & long reads sequencing samples must include a DNA size-ladder marker of at least above 48.5 Kbp (e.g. Biolab cat# N0341S), it is recommended to use Pulsed field gel electrophoresis.

In case you have chosen a different method or equipment, please state the name of the method and the manufacturer of the equipment.

It is to confirm, that the DNA samples, for which this COA is provided, were extracted in compliance with all instructions stated in NRGene standards and/or other requirements provided by NRGene and refer to the organism which the characteristics stated in the above table.

Prepared by: Click or tap here to enter text.	Approved by: Click or tap here to enter text.

Signature: ___________________	Date: Click or tap to enter a date.

DNA total mass = Volume*DNA concentration

Approximation: 10[µg/Gbp]*Haploid genome size[Gbp]; please consult NRGene Genomic Analysis services for genomes larger than 10Gbp

For genomes larger than 10Gbp, please consult NRGene Genomic Analysis services.

Ideally use seedlings that have been dark treated for 24-72 hours

Refer to NRGene standard for information regarding the required/recommended methods and equipment.