DNA Sequencing FAQ
How do I set up a new account with 1st BASE?
Who should I contact?
You can email or contact us as below:
+65 6775 7318
Contact our respective sales representative in your region
Contact our respective sales representative in your region
Do 1st BASE offer trials for the Sanger sequencing services?
We offer trials for new customers to try out our Sanger sequencing services. Please contact our sales representative in your region to find out more.
What is the cost for the services?
You will be able to view the price upon ordering on our 1st BASE Online Ordering system. If you are placing an order with 1st BASE for the first time, the pricing displayed might not be updated. Please do not worry as you can still proceed with the ordering. We will update the pricing accordingly as soon as we confirm your order.
Alternatively, please contact our respective sales representative in your region for pricing details.
Can I send orders outside of Singapore and Malaysia? How do I go about doing it?
Sequencing order collection points are set up in a few locations in Singapore and Malaysia regions. Please contact our respective sales representative to find out more or if you wish to set up a new collection point.
Sequencing order collection point available in most institute. Free local collection.
Kuala Lumpur/ Klang Valley: Our dispatch will collect at your location
Please contact the local distributor in your respective countries
Why should I choose 1st BASE?
1st BASE is the leading provider of Sanger sequencing services, offering high quality results at fast turnaround time. Our other competitive edge includes:
- Daily collection and processing of sequencing orders
- High quality, long read lengths with fast turnaround
- Email notification of sequencing results, electropherograms and sequences
- Prompt technical support and consultation
- Free consultation and troubleshooting of the failed reaction
ORDER PREPARATION AND SUBMISSION
What is the ordering process?
You can place an order with us online via 1oo or email.
Alternatively, you can refer to our quick guide for registration and ordering.
What is the difference between the different services? Which service should I choose?
We offer comprehensive service to meet your needs! You can choose the service based on your DNA template type. If you are unsure, kindly contact us.
Single Pass DNA sequencing
Standard Sanger sequencing service for purified DNA templates. Guaranteed read length of over 1000 bases for good quality DNA templates.
Unpurified DNA template sequencing
Complete Sanger sequencing solution from unpurified PCR and unpurified plasmid products. Guaranteed read length of over 1000 bases.
Sanger sequencing run of post-cycle sequencing product with or without the cleanup completed.
Difficult DNA template sequencing
Optimized protocol developed for template with secondary structures, large size, high GC/AT content and other inherent factors.
Direct Colony Sequencing
Direct sequencing of colonies for high copy number of plasmid from agar streaks. Guaranteed read length of over 600 bases.
What kind of DNA templates are accepted?
We accept purified and unpurified DNA templates, in both tube and high throughput 96-well format
- Purified and unpurified PCR products
- Purified plasmids
- Post-cycle sequencing products before or after clean up
Please refer to the DNA template requirement guideline. Additional charges are applicable for DNA template purification prior to sequencing.
Please check with us for requirements for DNA template such as cut agar, bacterial culture, glycerol stock and colonies streak.
What is the Sanger sequencing workflow?
1st BASE DNA Sequencing Workflow
What are the quality check you have in your workflow?
A control sequencing reaction is performed with every run in our Genetic Analyzer. Sequencing reaction controls is used to evaluate the quality of the overall reactions. Our control reaction has to achieve Phred40 data and a minimum CRL of 1000 bases in order to meet our QC benchmark. We provide our control sequencing electropherogram in each of your sequencing order at no charge.
What is the DNA template requirement?
Please refer here.
How should I prepare the DNA template?
The quality of Sanger sequencing result is closely dependent on the purity and optimum amount of DNA template.
For plasmid DNA:
Most commercial DNA clean-up kits will give DNA of sufficient quality for Sanger sequencing. The most common problem arises from excess salts which usually occur if DNA was eluted in a high salt buffer or diluted/suspended in TE buffers. For Sanger sequencing, we recommend keeping the DNA in 10mM Tris-HCI pH8.0 buffer or deionized water. We provide plasmid extraction service as an add-on service.
For PCR products:
PCR clean-up is required to ensure that the DNA is free of contaminants, unused primers and dNTPs. Unpurified PCR products are not suitable for sequencing as it will compromise the quality of the sequencing result. It is also highly recommended that an agarose gel is run to verify that the PCR products is single band with correct target size. Purification can be done using any commercially available kits for gel extraction or PCR clean-up. We provide PCR clean-up and gel extraction service as an add-on service.
For Ready-to-load (RTL) reactions:
Both cycle sequencing reaction setup and post-cycle sequencing clean-up are equally crucial for quality of DNA sequencing results.
We accept reactions performed with Applied Biosystems BigDye® Terminator Cycle Sequencing Kit v3.1. If other sequencing chemistry kit is used, please enquire.
We recommend the extension products of BigDye® reactions or cycle sequencing reactions to be purified immediately. If this cannot be done, freeze your product at -20°C upon removing them from thermocycler. Purification can be done using standard EDTA-Sodium Acetate-Ethanol Precipitation (as recommended by Applied Biosystems for BigDye® Terminator Cycle Sequencing Kit v3.1). Other commercial post-sequencing clean-up methods that are available in the market include column/ plate purification using gel bed filtration, magnetic beads, ethanol precipitation with co-precipitant and etc. As long as there is no carry-over of ethanol residue or water after the drying process, the purified cycle sequencing reactions should be readily load into Genetic Analyzer. For cycle sequencing reactions purified by magnetic beads, please indicate the elution volume (by water). We provide ready-to-load sequencing reaction with clean-up service.
Can I sequence unpurified PCR products?
If the residues of the PCR primers were depleted after the PCR, the noise background generated during the cycle sequencing may not affect the major base calls. However, the presence of the other contaminants (e.g. dNTPs, salts and etc.) could still compromise the quality of the DNA sequencing result to some extents.
We provide PCR clean-up service as an add-on service.
Do I need to lyophilize the DNA? Do I need to keep my DNA template is 4oC?
It is not necessary to lyophilize or dry your DNA templates. Please submit your DNA templates in deionized water at room temperature. DNA is stable in water at room temperature for a few days.
Should I quantify my DNA template before sending for sequencing?
The quality of DNA template is the most common issue in problematic Sanger sequencing reads. The amount of DNA templates used in sequencing reaction can affect the quality of data. Too little DNA template will generate low signal strength while too much DNA templates will generate a ski-slope effect which causes the signal to weakens gradually.
It is essential to accurately quantitate DNA templates prior to sequencing. However, accurate quantification of dsDNA is not easy.
It’s informative to run your DNA template on an agarose gel, using some standard of known concentration and estimate the relative fluorescent intensity following gel staining. DNA ladders/markers can be used as a reference standard since it comes with a known amount of DNA (ng) for each band.
Why is your measured concentration different from mine?
Generally, there are 2 commonly used methods to quantitate DNA templates. But neither method will show the presence of contaminating salts.
- Agarose gel electrophoresis – Purified DNA should run as a single band on an agarose gel (Uncut plasmid DNA can run as 3 bands: supercoiled, nicked, and liner). Contaminating DNA or RNA will show up in the gel.
- UV Spectrophotometry - The A260/A280 ratio should be between 1.7 and 1.9. Smaller ratios usually indicate the presence of contaminating proteins or phenol. The NanoDrop ND-1000 spectrophotometer takes 1-2 µL sample volumes and can detect concentrations from 1.5 - 3700 ng/µl without dilutions.
Traditional absorbance invariably overestimates DNA. In a finding, Kapp et al (2014) encounter a five-fold difference between absorbance-based method and fluorescent-based method, which could theoretically result in using lower amounts of starting DNA for the downstream sequencing step and cause unsatisfactory results. Discrepancies between the two methods were also reported by Deben et al, O'Neill et al and Simbolo et al (2013). The measurements at 260nm (A260) fluctuate with contaminants and buffer component, mainly because miniprep kits on the market often inevitably introduce some RNA, chromosomal DNA, and other fluorescent cellular material during the purification process that will absorb UV light. These other chemicals won’t necessarily inhibit the sequencing reaction, but they will contribute to the A260 reading. As a result, relying on the spectrophotometer reading alone will cause you to add less DNA template than you think.
Fluorescence assays on the other hand are less prone to interference. Fluorescence-based quantitation is more sensitive and more specific for the nucleic acid of interest. In the technical note from Invitrogen, comparison between the UV absorbance method and the fluorescent method was compared. Nanodrop method was found to shown fluctuation particularly at lower concentration range. Whereas, fluorescence based quantitation generate accurate and precise result across lower concentration range.
Our lab adopts fluorescence-based measurement which might explain the difference in the measured concentration. We take every step to ensure that the quantification process is consistent and accurate for downstream processes. Our laboratory apparatus is calibrated and maintained regularly to ensure they are working within their specified limits.
Kapp JR, Diss T, Spicer J, et al Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial Journal of Clinical Pathology Published Online First: 27 November 2014. doi: 10.1136/jclinpath-2014-202644
Simbolo M, Gottardi M, Corbo V, et al. DNA qualification workflow for next generation sequencing of histopathological samples. PLoS ONE 2013;8:e62692
My DNA template shows insufficient amount, can I still send it for sequencing?
We do not recommend sending DNA template of insufficient amount. Although a specialized protocol will be deployed to maximize the amplification, we are unable to guarantee optimal Sanger sequencing result.
Do you accept difficult DNA templates?
1st BASE continuously innovates and developed methods to overcome technical difficulties of Sanger technologies. Our in-house optimized protocols are developed to overcome sequencing of difficult DNA templates with secondary structures, large fragment size, high GC content, high AT content or long repeats region.
What is the minimum size of DNA fragment that you can sequence?
Ideally DNA templates should be 100bp and above. With our optimized running protocol and module, we can sequence DNA templates as small as 90-100bp, but this will be subjected to the DNA template and primer quality.
For DNA templates as small as 75bp, we can perform BigDye Terminator Cycle Sequencing Kit v1.1 at separate run and cost.
For DNA templates less than 75bp, we recommend for the sequencing to be done using next-generation sequencing.
What universal primers do you offer?
Please click here to view the list of universal primers we offer.
How long will my DNA template and sequencing primers be kept?
How can I reuse them for my next order?
We will only keep DNA templates and sequencing primers for 1 week upon receiving them. Please indicate in the order form if you would like us to keep your DNA templates or sequencing primers for further sequencing reactions. A nominal fee will apply for storing your DNA templates or sequencing primers in our laboratories.
When can I receive the result?
Upon the receipt of your sequencing order to our DNA sequencing facility, an order confirmation will be sent to you through email. We typically deliver the sequencing result to you within 48 working hours upon the receipt of your complete and accurate DNA sequencing order. For ready-to-load reactions, results are typically ready within 24 working hours.
How do I receive the result?
Results are downloadable through 1st BASE Online Ordering account or via an email with the password protected weblink to our server. The compressed file in .zip format (for Windows), contains the complete sequencing results with (i) electropherogram(s) in .ab1 file and (ii) fasta sequence in .seq file.
How safe is my result data?
We understand that the confidentiality of your result data is critical. To ensure a high level of security towards the result uploaded, we have provided result in email with password protected.
What are the software that can be used to view the result?
The electropherogram, saved as .ab1 file, can be opened and analyzed using various freeware: Chromas, FinchTV, Applied Biosystems Sequence Scanner 2.0 and etc.; while the fasta sequence file can be viewed using notepad.
How long is the result link valid?
The result stored in your online account has no expiry. You may download at your convenience via your 1oo online account.
For customers who do not have the 1oo online account, the web link provided in the email to download the results has a validity of 3 months.
My access code is invalid. How do I get a new access code?
Please contact the respective sales representative in your region so another access code can be provided to you via email.
I have encountered an error when I want to download my results online via 1st BASE Online Ordering account. What should I do?
Please contact the respective sales representative in your region so they can troubleshoot further.
General guideline for a good design of sequencing primer
Ideally, sequencing primer should be designed 100 bases upstream of your sequence of interest. Below are some other consideration when doing your primer design:
- Length of primer to be between 18-25 bases
- GC content to be between 40-60%
- Melting temperature at around 55-60°C
- Check for significant hairpins (>3bp) and homodimerization
- Choose your primer desalted
We require approximately 10 pmoles (or 1µl of 10µMolar) of primer per sequencing reaction. Please send 5 µl per reaction as some evaporation may occur.
Many commercial oligo providers offer online design tools which you can use. See the link to IDT’s OligoDesigner.
For enquiries on analyzing results and troubleshooting