Sanger Sequencing FAQs
Source BioScience can process the payment of sequencing orders through purchase order number, eVouchers or credit card.
Source BioScience offers a range of single read and 96 well plate eVouchers. Click on the following link to find out more and order your Sanger Sequencing eVouchers.
Each reaction that is ordered on the website requires one eVoucher to cover the cost of the reaction. When you get to the ‘your basket’ page on the order form simply input the number of eVouchers you are using on the job and click update cart in the bottom right-hand corner. This will subtract the cost of the eVouchers from the order. eVouchers are automatically deducted from your account. If you are submitting a full plate for sequencing then 96 single read eVouchers or 1x 96 well plate eVouchers will be required as payment.
Yes, you are now able to use specific e-vouchers for all Sanger pre-treatments and types of order. The full range of e-vouchers can be purchased here.
Our eVouchers page allows you to buy vouchers in single reaction or plate format for all the pre-treatments. These are purchased and added to your account, you are able to transfer these to other users in your organisation as well if you wanted one main purchaser. This means you no longer need a Purchase Order number to cover the extra.
The sample requirements for Sequencing can be found in the following link DNA and Primer Sample Requirements.
Yes, we now order stand-alone primers using this link. Primers can be purchased using e-vouchers or via a Purchase Order number.
Primers that are ordered through the website are stored for a year and can be selected from the drop-down box on the reaction details page.
You can select this as an option when placing your order. Select the most recent order/s as required from your available options and then continue to place your order as usual. Your previous samples/primers are available in the drop-down menu when you are selecting your reaction specifics for you to choose as required.
Yes, all primers we process need to be sent to us in separate tubes so that we can prepare the Master Mixes with the correct ratio of reagents and primer.
Yes, although we require primers sent at different concentrations for the Colony PCR and Sequencing. Our primer requirements can be found here.
You can check what primers Source BioScience stock by clicking here.
If you are unable to generate the volume and concentration of DNA we require then please contact the laboratory or our sales team who can advise you whether it is worth sending the samples. The sequencing reactions can be optimised to work with stronger/weaker DNA.
In Sanger Sequencing we offer the following reaction types and pre-treatments, all are available to be purchased as eVouchers for your convenience.
Standard processing of a Plasmid sample through Sanger Sequencing.
A pre-treatment for Plasmid samples only, this service involves a 12hour incubation period to increase DNA concentration and remove any inhibitors.
Standard processing of a PCR sample through Sanger Sequencing.
A pre-treatment for PCR samples only. The submitted samples are purified prior to processing to remove any excess dyes/salts or other inhibitors that are present that can interfere with the reaction.
This service is for processing prepared samples where they have already been combined with the primer in the required quantities and concentrations. Please note no repeats are able to be processed on these reactions as we are unable to optimise the reaction conditions.
Sanger Run Only
This service is where customers supply a purified sequencing reaction that is ready for processing on the sequencer. Their DNA and primer have been PCR cycled with a dye terminator kit and subsequent purification has been processed.
Sanger Run Only Clean-up
This is a pre-treatment service for Sanger Run Only samples where the DNA and primer have been PCR cycled with the dye terminator kit and the reactions simply require the final purification prior to running on the sequencer.
This service is for when we are required to extract the plasmid from a cell pellet or liquid culture that is submitted prior to sequencing.
This is a fragment amplification service where we generate a PCR product using submitted primers prior to processing purifying and sequencing the sample.
Analysis Settings and Data Options
We aim to return all Colony PCR data within 24 hours of sample receipt.
The Quality Value Limit is the threshold setting to which bases will be scored accurately. If a QV setting of 10 is selected, the base caller will assign a base call to each peak it recognises and gives a confidence score with each base, blue being very confident, yellow is relatively confident and red is less confident. If the base caller cannot identify a valid base an N will be assigned. If a QV setting of 20 or 30 is selected, the base calling becomes more stringent, removing the yellow and red confidence bars and only offering blue confident base calls or N base calls.
Mixed Base Identification
Mixed bases are one-base positions that contain two or more bases. If the mixed base identification is selected the base caller assigns an IUB code to every base that contains a second peak at the same position which is >25% of the largest peak. If the mixed base identification is not selected, the base caller assigns an A, C, G or T to every peak. Bases that have a quality value < than the QV setting selected (10, 20 or 30) will be assigned an N.
A – Adenine
C – Cytosine
G - Guanine
T - Thymine
B – C or G or T
D – A or G or T
H – A or C or T
V – A or C or G
R – A or G
Y – C or T
K – G or T
M – A or C
S – G or C
W – A or T
N – any base
The start and end of a sequence read can be difficult for the base caller to accurately assign bases to the peaks. The Quality Clipping setting is the removal of the start and end base calls until fewer than a set number of bases have a QV setting of less than the default.
We keep the Colony PCR samples and primers for the standard 3 weeks before discarding them. If you would like us to store them for longer then please contact us to request this (firstname.lastname@example.org)
For an in-depth description of the PeakTrace™ Basecaller, please click here.
Source BioScience provides a chemistry known as the dGTP chemistry to help resolve sequencing issues such as G-C rich templates, hairpin structures, repetitive regions or troublesome sequences. The dGTP chemistry can be selected on the online order form.
For an in-depth description of the quality value limit, mixed base identification and quality clipping selections then please click here.
Each sequence reaction will produce 3 files in different formats. These are the .ab1 file, the .seq file and a .phd file.
The .ab1 file contains the electropherogram and raw data traces and can be opened in most sequencing software, click here to be directed to a website for downloading the free software FinchTV.
The .seq file contains the base calls from the sequencing reactions which can be opened in notepad, word pad or word document.
For a detailed description of the different types of sequencing traces generated please click here.
Additional email addresses can be added at the bottom of the ‘Prepare your samples’ page on the online order form. If you are inputting more than one email address please separate with a semi-colon (;).
General Sequencing Order Queries
No action is required, our collection points are visited whether an order has been selected for pick-up from that site or not, so we will still receive the order as normal.
We may need to be made aware of Adhoc samples sent to our collection points.
Due to the fast turnaround in which we process reactions, unfortunately we are unable to accept alterations to orders. If your order has not yet been received, you are able to cancel it via your Order History and place a replacement order. Please email the lab to confirm the new order number to avoid delays with your reactions.
We offer a repeat sequencing service for failed runs from samples which meet our submission requirements. If you receive reads that fail to generate sequences as per your expectations, please reply to the email within which you received the data and list the reaction(s) that you would like repeated. Any background information on size of the fragment and sample type can help us troubleshoot the data. Our lab team will analyse the submitted samples to verify that the sample requirements have been met. If the samples are found to meet our requirements, the sequencing will be repeated, free of charge.
Should your samples fail to meet the requirements, Source BioScience will offer a courtesy repeat sequencing service on up to 10% of the samples, to see if the deficiencies can be adjusted for. You will be made aware of the failed reads immediately and will be given the option to determine which samples, if any, will undergo the repeated sequencing.
You can contact us here to request the repeats you require.
You can contact the lab that processed your reactions by either replying to the data email that contained your data files or contacting the lab using the contact details at the bottom of the data email.
The website has a limit of 12 characters for sample names and primer names, so if the names you are entering are longer, the website will not allow you to proceed with the order.
You can only input certain characters on the website, so please use only underscores (_), letters and numbers.
Full stops (.), dashes (-), brackets (), slashes (/), stars (*), question marks (?), hash symbols (#) and commas (,) are not permitted on the order form.
Contact us today and one of our skilled account managers will be in touch with a free consultation including further information and pricing details.
Established as a market-leading Sanger Sequencing service provider, we deliver rapid turnaround times for sequencing using our streamlined SpeedREAD™ data delivery system, to the highest quality.
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