The preparation of various samples for ordering a sequencing service depends on the type of sample being submitted, the type of sequencing chemistry being ordered and the additional services being requested. Prepare your samples based on the guidelines below.

I. Concentration

Prepare stock solutions of purified/prepared templates and primers in ddH₂O based on the size of the template being sequenced. Use these stock solutions to  assemble samples to be submitted in tubes or plates. These guidelines do not apply to samples for Ready2Load Sequencing service.

Table 1: Concentrations of the stock solutions of primers and prepared templates

Note that the concentration of the Template is in ng/μL while the concentration of Primers is in pmol/μL.

II. Volumes

The volumes of the Sample stock solution(s) and the Primer solution(s) depend on the type of the sequencing services required. Ensure that the stock solutions are prepared according to the guidelines in table 1 before proceeding to section below.

1. 'Premixed' Samples:

When you submit your samples as 'premixed', ensure that the primer is mixed with your purified/prepared template. After assembling the Templates and Primers as described in the table 1 and prepare your sample by mixing the following volumes of the two stock solutions.

Note that only the premixed samples must be submitted for ShortSeq plate and the SimpleSeq Premixed sequencing services. Ensure that the samples for 'ShortSeq plates' or 'SimpleSeq Kits - Premixed' are prepared based on the guidelines set up here for 'Premixed' samples.

2. 'Templates Only' Samples

When you do not have the standard or custom primers handy or ready for use, you can submit 'Templates Only' Samples. For the primer required in this sequencing reaction, you can

• Use one of our >75 universal primers to be added to your sample before sequencing (free), or
• Order the synthesis of a custom primer that will then be used for sequencing your samples and subsequently stored in our facility for your future sequencing orders

Table 3: Assembling 'Templates Only' Samples.

3. 'Templates and Primers' Samples:

When you submit 'Templates and Primers' Samples, our sequencing technicians will mix the primer(s) with your sample(s) prior to sequencing. The ability to send primers and templates separately allows you to submit samples for multiple sequencing reactions or order additional services such as PCR purification/DNA Preparation for your template sample. The following table provides guidelines for the submission of prepared templates and primers. Use the concentration of templates and primers in table 1 to prepare the stock solutions. When you submit 'Templates and Primers' samples:  

• You must submit your primers and templates in separate containers,
• Concentrations of template DNA and Primers must adhere to the recommended values (see table 1).
• Provide sufficient quantities of samples and primers (see table below). You can order multiple reactions for a template submitted in 1 tube or a single well of the 96-well plate. Please note that the volume requirements vary for standard and power read sequencing services.
• Use barcode labels (optional) on the primer and sample containers,
• Indicated on the tube sequencing (check box) or plate sequencing (mention in notes) order editor if you want the sent primer to be stored at our facility for your future orders. On request, these primers will be stored in our facility for 1 month or till they are exhausted, which ever is earlier.

Table 4: Assembling 'Templates and Primers' samples

III. Samples Requiring 'Additional Services':

When your sample requires further preparation or purification prior to sequencing, the following conditions should be met:

• The template(s)/Sample(s) and primers(when enclosed) should be submitted in separate containers
• The volume of the sample based on its type should meet the following requirements (see table).

Table 5: Assembling samples which require additional services

How to Label Colonies

Prep Information

• Shortly centrifuge the received plate to avoid the liquid on the foil. Carefully remove the sealing foil from the plate
• Grow your colonies on agar plates long enough to have a colony diameter of at least 1 mm*
• Use a toothpick to take as much of the bacterial colony as possible and inoculate into our plates. Swirl the toothpick for some seconds to transfer as many cells as possible into the wells
• In case of bacterial suspension use a pipette to add 5µl of the liquid culture in each well
• Well H12 should be kept empty for internal quality control
• Seal your plates using 8-cap strips to prevent material loss (8-cap strips are provided along with each PlateSeq Kit Colony)
• Plates can be sent / provided at ambient temperature to our sequencing lab

Volume and Concentration

Primers

Thanks to our DNA synthesis lab, Eurofins Genomics is able to synthesize and stock primers in-house. This offers greater flexibility to our customers when it comes to picking a sequenicng primer.

Standard (universal) primers - free
Select from over 80 free, standard primers.

Custom synthesized primers
Specific primers can be ordered directly with your sequencing order. These primers are stored for 4 weeks and can be re-used during this time

Send your own primer (enclosed)
You can send us your sequencing primers in 1.5ml tubes along with your samples by using our free barcodes.

Optimum primer conditions:

• Primers must not contain phosphorylation or fluorescent dyes
• The optimum primer length is between 16-25 bases
• The primer melting temperature (Tm) should be 50 - 62°C
• The GC content of the primer should be 35-60%
• Ideally one G or C should be located at the 3' primer end
• The number of 3' Gs or Cs should not exceed 2 Gs or Cs
• If possible, avoid >3 identical bases in a row in the sequence

Primer concentration and volume:

• Exactly 10 pmol/µl primer concentration is required per sequencing reaction
• Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl); 5 µl of primer volume is required for every additional sequencing reaction
• Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer

n = number of bases within a wobble according to IUPC code, X = number of wobbles within the primer sequence. [e.g. 1 V (AGC) = 31 x 10 pmol/µl; 2 V (AGC) (AGC) = 32 x 10 pmol/µl]

*: Purchased Tube Labels can still be used to identify enclosed primers

Failures

Definition of technical failure
A technical failure is defined as a chemical or technical (IT, machines) defect in our lab. In order to ensure that the sequencing quality is not affected by a technical failure, an internal quality is run with each plate (for that purpose well H12 should be kept empty, otherwise a quality control is not possible). In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths. If these parameters fall below a certain percentage across the plate an additional manual check is performed. If a technical failure is confirmed by this manual check, the plate is getting re-sequenced.

When your sample requires further preparation or purification prior to sequencing, the following conditions should be met:

• The template(s)/Sample(s) and primers(when enclosed) should be submitted in separate containers
• The volume of the sample based on its type should meet the following requirements (see table).

For customers who need to store their sample(s) before submitting it, we recommend storing both samples and primers in a freezer.

Note that you can provide the details of the enclosed primer (Send a primer; primer enclosed in a separate tube in the shipment) or order the synthesis of a new primer through the controls on the left. To order/send two or more primer syntheses, simply hit 'add primer to list' after each entry; these primers will automatically be added to the my favorite primers dropdown list. You can manage the primers displayed in the dropdown box by removing/adding the primers from/to the 'My favorite primers' box on the sequencing dashboard. Please note that the ordered and sent primers will be stored for future use in your sequencing reactions. You can either choose to have these primer stored exclusively for your use or share the same with your team by including them in your group using the group tool.

How to download results?

Go to your Order History. Next, click the order to expand and reveal the download link. Click the link to download a zip file of the results. To visit the Order History page, click one of the many links located throughout the website, including in the header beside your account name.

When looking at the order history page, each type of order is color coded by its product line, so it is easy to find the order you want. For example, sequencing orders are marked by a small blue icon, oligo orders are marked by an orange icon, and so forth. Please note that sequencing results are only saved for a limited amount of time. If you are trying to download very old results, it may not be available online but may be archived and available by manual retrieval. Contact us for more information.

Selecting Preferred File Types

Users can select which file types to receive during the "Preferences" step of checkout. The file types can different between the sequencing method and file type. Simply check the box beside the desired file types to select them. The following screenshot shows the file types available for Sanger sequencing. 

Please visit the shipping samples page for all information on physically submitting samples. *It is highly recommended to print the order confirmation page and include it with your samples. This ensures the fastest processing time. 

I. Overnight Sequencing Guidelines

Overnight Sequencing service from Eurofins Genomics allows you to advance your research project in an uninterrupted manner within a limited budget. To avail the Overnight Sequencing service from Eurofins Genomics, your order/samples must meet the following requirements:

• The samples are shipped using 'Overnight Shipping' service
• The order must require less than 192 sequencing reactions
• This services applies only to samples requiring 'Standard Read' sequencing chemistry (this does not include other services such as power reads, BAC, PCR purification or RCA)
• Samples must be supplied in one of the following containers
  • 1.5-mL or 2-mL centrifuge tubes with barcode labels* pasted on them
  • 96-well plates with a free barcode label pasted at the bottom of the plate
  • Use barcode-labeled tubes or semi-skirt Plates supplied by Eurofins Genomics, e.g., as tubes from SimpleSeq Kits or 96-well plates from the PrePaid plate Kits
  • Use of Screw Caps or Sealing using parafilms prevents cross contamination, but can extend the turnaround time.

II. Ready to Load Sample Guide

Ready2Load sequencing services is provided for samples that have a single requirement: to be run on the sequencing gel. Samples being submitted as Ready2Load samples must meet the following requirements:

• The BigDye v3.1 reaction must be completed.
• The purification steps that remove unincorporated dye molecules must have been completed.
• It is recommended that these samples are sent as dry pellets.

Note that the Ready2Load plates are processed using an abbreviated protocol which typically have a read length of 500–700 bp.

III. Direct Colony Sequencing Guide

General Prep

• Shortly centrifuge the received plate to avoid the liquid on the foil. Carefully remove the sealing foil from the plate
• Grow your colonies on agar plates long enough to have a colony diameter of at least 1 mm*
• Use a toothpick to take as much of the bacterial colony as possible and inoculate into our plates. Swirl the toothpick for some seconds to transfer as many cells as possible into the wells
• In case of bacterial suspension use a pipette to add 5µl of the liquid culture in each well
• Well H12 should be kept empty for internal quality control
• Seal your plates using 8-cap strips to prevent material loss (8-cap strips are provided along with each PlateSeq Kit Colony)
• Plates can be sent / provided at ambient temperature to our sequencing lab

Sample Specific Prep

Volume and Concentration

Primers
Thanks to our DNA synthesis lab, Eurofins Genomics is able to synthesize and stock primers in-house. This offers greater flexibility to our customers when it comes to picking a sequencing primer. The three main types of primers are:

1. Standard (universal) primers - free.  Select from over 80 free, standard primers.
2. Custom synthesized primers. Specific primers can be ordered directly with your sequencing order. These primers are stored for 4 weeks and can be re-used during this time
3. Send your own primer (enclosed). You can send us your sequencing primers in 1.5ml tubes along with your samples by using our Primer Barcodes*

Please consider optimal primer conditions, concentration and volume as described below.

Optimum primer conditions:

• Primers must not contain phosphorylation or fluorescent dyes
• The optimum primer length is between 16-25 bases
• The primer melting temperature (Tm) should be 50 - 62°C
• The GC content of the primer should be 35-60%
• Ideally one G or C should be located at the 3' primer end
• The number of 3' Gs or Cs should not exceed 2 Gs or Cs
• If possible, avoid >3 identical bases in a row in the sequence

Primer concentration and volume:

• Exactly 10 pmol/µl primer concentration is required per sequencing reaction
• Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl); 5 µl of primer volume is required for every additional sequencing reaction
• Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer

n = number of bases within a wobble according to IUPC code, X = number of wobbles within the primer sequence. [e.g. 1 V (AGC) = 31 x 10 pmol/µl; 2 V (AGC) (AGC) = 32 x 10 pmol/µl]

*: Purchased Tube Labels can still be used to identify enclosed primers

Failures

Definition of technical failure
A technical failure is defined as a chemical or technical (IT, machines) defect in our lab. In order to ensure that the sequencing quality is not affected by a technical failure, an internal quality is run with each plate (for that purpose well H12 should be kept empty, otherwise a quality control is not possible). In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths. If these parameters fall below a certain percentage across the plate an additional manual check is performed. If a technical failure is confirmed by this manual check, the plate is getting re-sequenced.

When your sample requires further preparation or purification prior to sequencing, the following conditions should be met:

• The template(s)/Sample(s) and primers(when enclosed) should be submitted in separate containers
• The volume of the sample based on its type should meet the following requirements (see table).

IV. General Information on Sequencing Services and Sample Types

We have tailored our DNA sequencing services using the Sanger method considering the diverse natures of the projects and requirements of the researchers. Researchers can send different types of samples in different formats with or with out including the primers or requiring additional services on the samples.

Sequencing Services provided for various Sample Types based on the inclusion of Sequencing primers

Each of the sequencing reaction requires a Sequencing primer that is specific to the template being read. We provide the most widely used sequencing primers for no additional cost. However, if you do need custom primers, our DNA synthesis lab will synthesize and deliver the same to the sequencing lab with remarkable turnaround times.

#For more details on Next Generation Sequencing Services, click here.
*Additional charges apply.