FAQs
Answers to the most common questions about our Sanger Sequencing and Next Generation Sequencing services.
Table of Contents
Sanger Sequencing
Getting Started
You may create an online account by clicking on the ‘Sign In’ tab in the top right corner of this site.
Once you are in the Sign In page, click ‘Register Here.’ You will be asked to submit basic information such as your name, email address, institution, address, and phone number. You will also be prompted to create a password to use to log in.
After you have entered the required information, an account will be created and you will be able to place orders online.
Yes, we offer a free trial for the first order of 5 Single Pass Primer Extension reactions with free shipping. On our home page, click the Free Sequencing Trial. You will then be asked to enter your sample information including the names of your templates and primers, as well as the type of DNA you wish to have sequenced, which can be either plasmid or purified PCR products.
After entering your sample information, you will be prompted to open an account, where your order information will be stored and data will be uploaded.
If you are at an institution in which there is an active ACGT drop box, please place your samples in the drop box before the scheduled pick-up time. The samples will be collected by our courier and shipped to the designated ACGT facility. If your institution does not have a drop box, please email us at [email protected] to request a prepaid FedEx shipping label so that you may send your samples free of charge.
First, you will need to create an online account. Once you have done so, you can sign in to view your account dashboard. This page will have all of the services that are available to order online, so you will simply select the service you wish to order and fill out the order form. After submitting the order, you will receive an order confirmation number to include with your samples.
ACGT retains all account records on file. However, if you have not used our services since 2006, you must create a new account prior to placing an order. For all other users, sign in to your account as before. Once you sign in, click on ‘New Orders.’ From there you will be able to place an order for the service of your choice.
Choosing a Service
Low Cost Option (LCO): For premixed samples. Templates and primers must be mixed together in a single tube according to the concentrations specified in our sample submission guidelines. Banked primers cannot be requested for this service.
Standard: For samples of known concentrations. Templates and primers must be submitted in separate tubes, with concentrations clearly specified on the order form. Our technologists will use these values to optimize sequencing reactions.
Premium: For samples of unknown concentrations. Templates and primers must be submitted in separate tubes. A subset of templates and primers will be assessed via nanodrop and/or agarose gel to determine concentrations and optimize sequencing reactions.
For all of these services, ACGT universal primers can be added at no additional cost. You will simply write the name of the requested primer in that section on the order form.
Single Pass Primer Extension (SPPE), will provide individual traces for each Sanger sequencing reaction. Each reaction will produce up to 750-1000 bps of data, depending on the type of DNA. Consensus sequences can also be provided at an additional cost, if required.
Full Sequencing Analysis (FSA), is usually performed when the insert size is greater than 1.5 kb and requires additional assembly and analysis. For longer templates, the primer walking method is utilized to cover the full length of the sequence. Typically, a consensus sequence, alignment file, and a report outlining the results and variances will be sent to you if a reference sequence is provided. If you are not interested in receiving a detailed report, please use our SPPE service instead.
This depends on what your exact needs are. If you just need individual sequences that you can align yourself, you may use our Single Pass Primer Extension service. Our SPPE service can also provide you with the consensus sequence at an additional charge. Please make sure this option is selected on the online order form.
However, if you need a detailed report on sample processing, data analysis and variant report, please select our Full Sequencing Analysis (FSA) service.
You could choose either our Single Pass Primer Extension (SPPE) service or Full Sequence Analysis (FSA) based on the deliverables you would want. Please see the response to the question above for more detail.
Yes, ACGT can sequence such samples using one of our alternate protocols for difficult templates. Make sure to select either our High GC or Hairpin protocol under the Reaction column when placing your order.
Please note that additional charges may apply for these protocols.
Direct Colony Sequencing Service
We do not recommend suspending the colonies/culture in water as this can cause unfavorable sequencing results. We suggest using PBS pH 7.2 to 7.5, or TES (TrisHCl 10mM, EDTA 1mM, NaCl 150mM) pH 7.2 to 7.5 for sample suspension.
Initial results are typically made available 2-3 business days from sample receipt. Orders that require additional troubleshooting may result in longer turnaround times.
High Throughput Sanger Sequencing Service
Sample Submission
We recommend naming your samples in a simple, unique way. Long names are often difficult to write on a tube and abbreviations of these names can lead to human error. Our system will also only accept 23 characters for the combined template and primer name, including numbers at the beginning of the template. Please make sure that the name on the tube accurately and completely matches the name on the order form.
It is also important to keep in mind that our sample analysis software only accepts letters, numbers, underscores (_) and dashes (-) in the sample names. You must avoid using any other symbols in the names of your samples.
The sample names should be written on the top of the tube of the tube in black permanent marker. Please avoid using other colors as these are often difficult to read.
We recommend that you determine the DNA concentration by running it on an agarose gel and comparing it to a standard of known concentration, or using nanodrop.
We highly recommend purifying your DNA before submitting it for sequencing. Unpurified DNA often contains salts and other contaminants that may adversely affect the sequencing reads.
Column purification is sufficient for plasmids and PCR products with one single clear band. For PCR products that contain multiple bands, gel purification of the band with the desired size is required. We recommend following gel purification with a round of column purification. Further note, we recommend eluting the samples in nuclease free water instead of the elution buffer provided with the kit.
ACGT can also perform column or gel purification of your sample at an additional charge. Unpurified PCR products can be submitted under the SPPE Premium service.
Templates and primers are stored for two weeks after the order has been completed.
ACGT offers free banking services, which can allow you to store your templates and/or primers at our facility for use in future orders. Templates can be saved for up to two months, and primers can be saved for up to two years.
Please indicate on your order form if you would like ACGT to bank your samples.
Please see the instructional video below. A Sanger sequencing group member, Erin Murphy, presents tips on how to prepare your samples for high quality DNA sequencing data results.
Yes, you can ship your samples for delivery on Saturday. The Wheeling branch of ACGT is open on Saturdays for sample receipt and processing. However, there is no data delivery on weekends, so data will be uploaded on Monday.
DNA Sequencing Results
When you download the data, you will receive two files – a .seq file and a .ab1 file. The .seq file is a text file that can be opened using a word processor such as Notepad or Microsoft Word.
The .ab1 file is a chromatogram and may be opened using a variety of programs. If you do not currently have a chromatogram viewer, you can go to our Resources page to see a list of the available programs.
This will depend on the turnaround time you have selected and which type of order you have placed.
You will received an email notification once the data has been uploaded to your account.
PCR amplification primers may not always work well as sequencing primers.
Check that the parameters for your PCR primer design are suitable for sequencing as well. For sequencing primers, we recommend that they be around 18-23 bases in length with a Tm of 56-62 degrees. GC content should be between 40 and 60%. Primers with degenerative bases should not be used, as the primer should match the template exactly.
If you are still having issues with primer binding, it is possible that the PCR products generated may have lost the “primed” region. In other words, the region of DNA that the primer binds to may not be present in the PCR product. In order to overcome this, we advise using nested primers or designing new internal sequencing primers.
Double peaks are most often caused by primers binding in multiple locations or presence of multiple PCR products, but may also be caused by repetitive bases.
If you suspect that your primer is binding at more than one site, we would recommend using a different one; either a primer that is more specific or that will read the template from the opposite direction.
If you suspect that there is contamination and presence of multiple products, we recommend running your samples on a gel and ensuring that there is only one clear band. Multiple bands may mean multiple PCR products. If you are seeing more than one band, you may need to perform gel extraction on the band you wish to sequence.
For cases with repetitive sequences, we would recommend sequencing from the opposite direction and aligning the data.
The total cost of the order is dependent on the number of reactions performed by our technologists in the initial run, regardless of the quality of data returned. Any repeated reactions are performed as a courtesy and may be selected for re-sequencing based upon the analyst’s judgement when reviewing the results of the initial run. There is no additional charge for repeated reactions. Please see our repeat policy for more details depending on service type selected.
Miscellaneous
Samples and primers are normally stored for two weeks after they have been received. ACGT offers a primer banking service which allows you to store your primers and/or samples at our facility. Please indicate on your order form if you would like to take use this service.
Next Generation Sequencing
Description
Next-Generation Sequencing (NGS) is a high-throughput method used to determine the sequence of nucleotides in DNA or RNA. It enables rapid sequencing of entire genomes or targeted regions, facilitating applications in genomics, personalized medicine, diagnostics, and research.
We offer a comprehensive range of short (Illumina) and long (Oxford Nanopore) read NGS services, including whole genome sequencing (WGS), whole exome sequencing (WES), targeted gene sequencing, RNA sequencing (RNA-Seq), epigenetic sequencing, microbiome analysis, and many more. Additionally, we provide bioinformatics support and data analysis services.
- Read Length: Short Read NGS produces shorter sequences (50-300 bp) compared to Long Read NGS, which generates longer sequences (10,000+ bp).
- Accuracy: Short reads generally have higher per-base accuracy, while long reads may have lower individual read accuracy but can span complex regions.
- Throughput: Short Read technologies typically offer higher throughput, generating more data in a shorter time.
- Cost: Short Read sequencing is often more cost-effective per base, whereas Long Read sequencing can be more expensive but provides unique advantages.
- Applications: Short Read is ideal for applications requiring high accuracy and depth, such as SNP detection and gene expression profiling. Long Read is better suited for structural variant analysis, de novo genome assembly, and resolving repetitive regions.
How to Order
Placing an order is simple. All services available for direct ordering can be chosen from your account, and you can submit your project request by filling out an order form available on our website. For custom projects, contact our sales team directly. We will guide you through the process, including sample submission, service selection, and pricing.
Sample Submission
We accept various sample types, including blood, saliva, tissue biopsies, formalin-fixed paraffin-embedded (FFPE) samples, cell lines, and purified DNA/RNA. Specific requirements may vary depending on the service, so please refer to our sample submission guidelines or contact our support team for assistance.
We provide sample submission guidelines on our website, outlining necessary quantities and concentrations, shipping conditions, and packaging instructions. If you have any questions, our support team is available to guide you through the process.
Turnaround Times
Turnaround time depends on the type, scope and complexity of the sequencing service. Generally:
- Plasmids / PCR products / ready to load libraries: 1-2 weeks
- WGS / WES: 4-6 weeks
- RNA-Seq: 4-5 weeks
Expedited services are available upon request for an additional fee.
Costs
Pricing varies based on the specific service, sample type, and additional requirements such as data analysis. Please visit our website service pages for directly offered service prices, or contact our customer service team for a personalized quote tailored to your project needs.
Data Management
Yes, we offer comprehensive bioinformatics analysis services, either as an NGS project packages or as stand alone services. They include data processing, variant calling, annotation, and interpretation. Our team of experts can deliver customized reports based on your project requirements, whether for research, regulatory agency submissions, or other applications.
Absolutely. We offer secure data storage solutions and can assist with data management strategies to ensure your sequencing data is safely stored and easily accessible. Please contact our support team to discuss your specific needs.
Quality Management
We adhere to strict quality control protocols at every step of the sequencing process, including sample validation, library preparation QC, sequencing quality metrics, and data integrity checks. Our laboratory is GLP/cGMP qualified, ensuring compliance with industry standards for accuracy and reliability.
We prioritize data security and confidentiality. All client data is stored on secure servers located in our Wheeling IL facility, with restricted access, and we comply with relevant data protection regulations such as HIPAA. Additionally, we offer data encryption and secure transfer options to protect your sensitive information.
Yes, our laboratory operates in compliance with industry and regulatory standards, including CLIA (if applicable), and other relevant guidelines. We adhere to best practices in laboratory procedures, data handling, and reporting to ensure the highest level of compliance and quality.
Miscellaneous
Yes, we provide expert consultation services to help you design your experiments, select the appropriate sequencing platform and service, and plan your project to achieve optimal results. Contact our consulting team to schedule a session.
Yes, we provide metagenomic and microbiome sequencing services to analyze complex microbial communities. Our services include sample processing, sequencing, data analysis, and interpretation to help you understand microbial diversity, function, and dynamics in various environments.