Frequently Asked Questions

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Frequently Asked Questions About
Sanger Sequencing

Introduction

What is EZ-seq?

EZ-seq is a DNA sequencing service developed by Macrogen Europe. It uses 2D Bar-code labels for simple and secure tracking of the samples. It provides DNA sequence data with high reliability with fast turn-around time at reasonable price.

How does it work?

When you make an order, we ship the EZ-seq labels to you via regular mail. All you need to do is to mix the template with sequencing primer you want to use, and ship the samples to our laboratory with EZ-seq labels on the samples.


By the time you make an order, we already have your email address. So the EZ-seq labels issued are automatially linked to your email address, and any sequence data came from the sample with the labels will be directed to your email address. This means that you do not need to log in or fill the complicated order form everytime you have something to sequence.


Introduction about EZ-seq System

Why do I need it?

Have you ever felt bored of making long lines of order sheet with template and matching primer?
Have you ever been annoyed with so many invoices that came to you every time you send sequencing samples?
Have you ever wondered how much reasonably it was charged for your sequencing service?
Have you ever wanted to have your DNA sequences back earlier?
Have you ever felt it annoying to log in the web site every time you check out the results?

If you have any of above experiences, it is worth giving a try to EZ-seq, because EZ-seq is designed to get rid of such inconveniences. We are quite sure that you will be surprised to see how EZ the DNA sequencing can be.

What are the differences between four services?

EZ-seq service has two options, Tube vs 96-well plate, and Purification vs purified template. By combination of these four options, four different services are available. EZ-seq Purification service will go through PCR purification prior to sequencing.

Making order

Why do I have to provide all these information?

We tried to reduce the amount of information required, but found that there are some essential information without which we cannot properly deliver our service. So we beg your kind understanding for these. Once you've set up the account in our on-line system, you won't need to fill in those information again. Your information shall be encrypted and securely stored in our server, and shall not be used for any other purpose than correct delivery of our service.

Why is the email address so important?

In DNA sequencing service, Email address is just like delivery address of the final product. Let's say you orders one vial of Taq polymerase. The most important information in the order will be where the product should be delivered. So, having a proper and secure email address connected to the EZ-seq label is very much important.

Can I give my labels to my colleagues?

Whoever uses the labels, and wherever the samples with labels come from, the sequence data will be directed to the designated email address, so you will need to forward the results to your colleagues. For change of the recipient email address, please refer to the article right below.

Can I change the recipient email address from what was given by the time of order?

Change of delivery email address is the change of final beneficiary, and it is more or less like change of the contract, so it is our recommendation not to do this frequently. But we are quite a flexible in fitting ourselves to your needs, and we can do that for you. But we will need to get the written confirmation from both of two parties. Please contact us if you wish to proceed with this.

Is there any order sheet necessary?

No. You can make order from this web page, and there's no need for typing-in or importing the individual reaction information. You know better about your samples than we do, and we do not need to know what you are sequencing. All we do is to do our best with what we got from you!

Results

How long does it take until I receive the results?

EZ-seq is 24-hour service. From the moment of sample arrival, it will take between 12 hour ~ 24 hour depending on what time point the samples arrive. But we basically guarantee 24 hours if there's no problem with the sample condition.

What kind of sequence results do I get?

We provide the following files.

AB1 : Raw chromatogram file with quality score
SEQ : Sequence in plain text
PHD : Quality score file
PDF : AB1 files in PDF format for image processing or printing

They are compressed in ZIP file and the download link is sent in the result email.

How long can you read?

It depends on the sample condition, but in most ideal condition we read about 1,050 bases with Q20 or higher in more than 650 bases.

Payment

I have received the sequencing results, how can I process the payment?

Macrogen Europe will issue an invoice via email after data delivery. Invoices can be only paid by Credit Card (only VISA and Master) and wire transfer.

(*Ez-seq, Eco-seq and prepayment invoices are issued within 1~3 business days after the order placement)

I want to pay via wire transfer, what is your bank information?

Beneficiary Name: Macrogen Europe BV

IBAN: NL36 INGB 0008 8449 96

SWIFT Code: INGBNL2A

(* Please be sure to state the invoice number(s) in the description if you pay by wire transfer. Otherwise, your payment would take long to match our record.)

I placed several orders, can I receive only one invoice?

This should be discussed case by case since our billing procedures differ in order categories. Please directly contact us via info@macrogen-europe.com.

I have received an invoice and would like to change the billing information. What should I do?

Please contact our sales or invoicing department via info@macrogen-europe.com or invoice@macrogen-europe.com.

Why is my invoice charged 21% of VAT amount?

Macrogen Europe is applying to EU regulation such as Tax and Corporate law. Based on 1968 Sales Tax Act, ar. 12. 3 Wet OB 1968., we are obligated to charge our local VAT:

  • For customers from the Netherlands, 21% of the VAT amount will be added to the invoices.
  • For EU customers outside the Netherlands, 21% of the VAT amount will be added to the invoices if you don’t have a valid VAT number.

I would like to check whether my payment is well received. Who should I contact?

If you want to confirm the receipt of your payment or if you have any inquiries/issues regarding the payment you made, please send an email directly to ar.eu@macrogen-europe.com.

Sample condition

How do you do the purification of PCR products with primer already mixed?

Our R&D team successfully developed Macrogen's unique technology called MSE based on partial dephosphorylation, and could sepcifically eliminates the molecules with minority, and EZ-seq Purification adopts this technology. So please feel free to mix the primer.

I want to use one of the universal primers. Can I get this primer?

When you make order, you can tell use your preferred primer. We will send this primer along with your labels.

Can EZ-seq work on large-sized molecules like BAC or gDNA?

The success rate of sequencing reaction is in proportional to the number of molecules, not the amount of DNA. So even when we have same amout (ng or ug) of DNA, the number of molecules will be highly variable depending on the size of the DNA, that is, the smaller, the better. So generally the success rate of large molecules like BAC of gDNA is much lower than PCR products of plasmid. But by increasing the concentration of template DNA, we can match up the number of molecules to a certain degree. When you want to sequence these large molecules, we advise you to use the DNA with highest concentration possible.

What is the optimal concentration of DNA and primer?

In all cases, the volume of template and primer is 5ul + 5ul to make total of 10ul. The concentration varies depending on the services.

EZ-seq (without purification)
Template : 50~100ng / ul
Primer : 5pmole / ul

EZ-seq Purification
PCR product : 50ng/ul
Primer : 10pmole / ul

I'm in trouble!

I lost my labels.

Don’t worry. Your usage of label is kept in our LIMS(Laboratory Management Information System) server. Let us know of your situation and we will reissue the labels and send them to you. These labels will have same serial number with your previous labels, so it is important to discard the old labels even though you find them later as our LIMS validates every label by the time it is scanned.

I didn’t get the results yet.

There are three possibilities.

  1. Failure of sample delivery: Please provide us with the tracking number of your samples.
  2. Failure of sample processing: Due to some reasons (such as lid is open on the way, or no DNA in the tube) the samples may not be processed to save your labels. In this case, our staff may have contacted you via email. If you didn’t receive this email even in SPAM folder, please contact us immediately. (info@macrogen-europe.com)
  3. Failure of sample delivery: Please provide us with the tracking number of your samples.

Miscellaneous

How can you make the sequencing so inexpensive?

As many people know, the largest portion of sequencing cost is the dye terminator, machinery depreciation, and labor cost. Among these, cost saving in dye terminator already reached its saturation point, so the price comes from the other two. Thanks to the early adoption of our high-throughput sequencers, we already finished depreciation of the machines. Also by automation in the laboratory, the labor cost got much lowered. More than anything else, we are a scientists-dedicated company, and we keep our price to the level that we think is reasonable regardless of the market price.

Can I work in Macrogen?

The door of Macrogen is always open. If you have passion to become a global player in genomics field, please send your introduction with CV to us. Also, many internship possibilities are available. Please feel free to contact us.

Frequently Asked Questions About
Next Generation Sequencing

Introduction

What kind of NGS technology do you provide?

Download our NGS service brochure and check out our NGS Platform Suggestion page.

Is any promotion going on?

Check out our Blog/News page, or contact us via ngs@macrogen-europe.com

Can I see your bioinformatics analysis report?

Please request to Macrogen’s sales representatives for a demo report of your chosen application.

Are you GDPR compliant?

Yes, please check out our Privacy Policy and Data Security Policy.

What is your overall workflow?

Check out our NGS Sample Submission Guide page.

Order Placement & Shipping

I am happy with the quotation I have got. What’s the next step?

  1. Create your account at our ordering site dna.macrogen-europe.com. Billing information (name, address, email, phone, VAT number etc.) is required for the registration, so please prepare it in advance.
  2. Place your order by submitting 3 documents to Macrogen’s sales representative: 1) Purchase order approved by your affiliation (or credit card information), 2) Singed quotation, and 3) Filled-in order sheet
  3. Dispatch your samples via Macrogen’s online shipping system. Ask Macrogen’s sales representative for shipping instruction.  
  4. Once your samples arrive at Macrogen, NGS Sample Arrival Notice will be sent to your registered email.

For more info, please check out our NGS Sample Submission Guide page.

How can I ship my samples to Macrogen?

Contact your Macrogen representative to get access to our online shipment system. The following should be included in the package.

  • Prepared sample tubes
  • Adequate cooling agent e.g. ice pack, dry ice
  • Cushioning material e.g. bubble wrap, foam, tissue paper
  • Printed Macrogen order sheet

Print the 3x shipping labels and 3x commercial invoices, sign and enclose with your shipment and contact your local office of the chosen courier to schedule a pick-up and dispatch for the same day.

Your sample and data will be discarded after 3 months of data delivery. You may extend the period with a special request.

Sample Submission

How should I prepare my DNA samples?

Start by checking the requested input amount from our QC criteria.

DNA:

  • Quantify your sample using the fluorescent-based method.
  • Check quality of your sample by gel electrophoresis.
  • Aliquot your DNA sample in nuclease-free water or TE buffer in a clearly labeled 1.5~2.0ml microcentrifugetube sealed with parafilm tightly. Refer to our packing guideline.

Include a brief description of extraction and/or enrichment protocol used in the “Comments” fields of the order sheet.

What kind of a buffer should I use for my DNA samples?

Usually EB (Elution Buffer), DW (Distilled Water), TE (Tris-EDTA) are used, but if the buffer you are using is from a commercial kit, it is likely that is neutral PH hence would not affect further wet-lab procedures.

Certain carriers and pellet paint types are not compatible with aspects of our sequencing technology.

How should I prepare my RNA samples?

Start by checking the requested input amount from our QC criteria.

RNA:

  • Quantify your sample using the fluorescent-based method.
  • Check quality of your sample by BioAnalyzer or equivalent.
  • · The optimal condition for RNA storage and/or delivery is ethanol precipitation method (stable for 1 year at -20C) Alternatively, RNA transport reagents from various commercial companies can be used. e.g. Ethanol Precipitation
    1. All solutions should be nuclease-free or DEPC-treated water.
    2. Add 0.1 volume of 3 M Sodium Acetate (NaOAc, pH5.5) to RNA solution and mix gently.
    3. Add 2 volume of 100% ethanol to RNA solution and mix gently. If you have 100㎕ of RNA solution, add 10㎕ of 3 M Sodium Acetate (NaOAc, pH5.5) and 220㎕ of 100% ethanol to RNA solution and mix well gently.
  • Aliquot your RNA sample in nuclease free water in a clearly labeled 1.5~2.0ml microcentrifuge tube sealed with parafilm tightly. Refer to our packing guideline. If dry ice shipping is needed, please contact your Macrogen representative.

Include a brief description of extraction and/or enrichment protocol used in the “Comments” fields of the order sheet.

What kind of buffer should I use for my RNA samples?

Usually RNase-free water and DEPC-treated water are used, if the buffer you are using is from a commercial kit, it is likely that is neutral PH hence would not affect to further wet lab procedures.

If you are using RNA transport liquid or ethanol, please clearly state the buffer on our order sheet.

Non-robust results have been observed from samples where Lithium Chloride (LiCl), fluorescent pellet paint, or tRNA was used prior to precipitation and resuspension for sample preparation input.

How should I pack my samples?

Place sealed individual 1.5ml microcentrifuge tubes in a 50ml screw cap tube for additional insulation during shipment. To prevent sample tubes from moving during shipment, pack any remaining space in the 50ml tube with clean tissue paper prior to sealing. Refer to our packing guideline.

Contact your Macrogen representative to get access to our online shipment system. The following should be included in the package.

  • Prepared sample tubes
  • Adequate cooling agent e.g. ice pack, dry ice
  • Cushioning material e.g. bubble wrap, foam, tissue paper
  • Printed Macrogen order sheet

Quality Checks

DNA QC criteria

RNA QC criteria

Library QC criteria

If you have prepared and are going to submit a ready-to-run library for sequencing, please meet 5~10nM concentration and >15ul volume criteria.

Download DNA Library QC Criteria

Download RNA Library QC Criteria

Which method does Macrogen use for DNA QC?

Quantity of DNA

Macrogen quantifies the starting genomic material by a fluorescence-based quantification*, rather than a UV-spectrometer-based method.

This is because fluorescence-based methods, which employ a double-stranded DNA specific dye, will specifically and accurately quantitate dsDNA even in the presence of many common contaminants. UV spectrometer methods based on 260 OD readings are prone to overestimating the DNA concentration due to the presence of RNA and other contaminants commonly found in gDNA preparations.

* Picogreen (Invitrogen, cat.#P7589)

Purity of DNA

Absorbance measurements at 260 nm are commonly used to quantify DNA.

The ratio of absorbance at 260nm to absorbance at 280nm is used as an indication of sample purity, and values of 1.8-2.0 are considered indicative of relatively pure DNA.

Condition of DNA

Gel electrophoresis condition: 1% agarose gel, 30min running at 160V, 0.5ul of DNA loaded, with 1kb marker

Size checking of DNA [Upon request, Extra fee will be charged]

  • DNA fragments <1kb : 2100 Bioanalyzer** is used for checking the size.
    **Macrogen use DNA 1000 chip and DNA 7500 chip for normal PCR product, high sensitivity chip for very small amount of DNA fragment such as ChIPed DNA.
  • DNA fragments < 150kb : PFGE method is used for large size of DNA

Which method does Macrogen use for RNA QC?

Macrogen quantifies RNA samples by a fluorescence-based quantification. We also check their integrity with an RNA Integrity Number (RIN) value using an Agilent Technologies 2100 Bioanalyzer or equivalent.

RNA that has DNA contamination will result in an underestimation of the amount of RNA used. We recommend including a DNase step with the RNA isolation method. However, contaminant DNA would be removed during mRNA purification.

It is very important to use high-quality RNA as the starting material. Use of degraded RNA can result in low yield, over-representation of the 5' ends of the RNA molecules, or failure of the protocol.

Which method does Macrogen use for illumina library QC?

To verify the size of final library fragments, we check the template size distribution by running on an Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip.

In order to achieve the highest quality of data on Illumina sequencing platforms, it is important to create optimum cluster densities across every lane of every flow cell. This requires accurate quantitation of DNA library templates. So, we quantify prepared libraries using qPCR according to the Illumina qPCR Quantification Protocol Guide.

Data Analysis Result

How can I check the integrity of downloaded files?

MD5 checksum is used in order to verify the integrity of downloaded files. If the values of md5sum are the same, there is no forgery, modification or omission.

Download your result files from the secure FTP links, do not decompress them yet, check md5sum of the files, and compare with the provided ‘md5sum’ in the report file (pdf/html).

Payment

What is your payment term?

Macrogen will issue an invoice after data delivery and payment term is NET 30 days. If you have any question, please contact your Macrogen sales representative or ngs@macrogen-europe.com

Bank account of Macrogen Europe B.V.

Wire transfer to the bank account of Macrogen Europe B.V.

Beneficiary: Macrogen Europe B.V.

IBAN: NL36 INGB 0008 8449 96

Bank Info: ING bank

BIC: INGBNL2A

I have received an invoice, and would like to change billing address. What should I do?

Contact our invoicing department via invoice@macrogen-europe.com

I would like to check whether my payment is well received. Who should I contact?

If you want to confirm the receipt of your payment or if you have any inquiries/issues regarding the payment you made, please send an email directly to ar.eu@macrogen-europe.com.

Miscellaneous

Terms & Conditions

This terms and conditions are applicable to NGS orders placed from March 2015 onwards.

A. Sequencing raw data and its analyzed result will be discarded 3 months after the data delivery. A specific request for long term storage is to be negotiated separately.

B. Whilst we are willing to examine client queries surrounding the data, we dictate that any formal objections should be addressed within 3 months after the data delivery.

C. Processed sample and any unused sample (back-up, QC failed one, etc.) will be discarded 3 months after the data delivery, unless otherwise special request is made.

D. At request, the samples can be returned at the recipient’s cost. Request should be made no later than 1 month after the data delivery. Usual shipping charge is $100 to $200 depending on the region. For the project amount of total $15,000 or above, the shipping and handling charge can be waived.

E. During data storage period, analysis result of your sample might be used for sample quality check process, data validation, internal education or other investigation for better service by Macrogen.