DNA Sequencing

 

How do I go about using the Source BioScience LifeSciences DNA sequencing?

These are the minimum things you need to do:

  • get your sequencing template prepared by PCR or plasmid extraction.
  • work out what primers you want to use for your sequencing; are they in our list of standard primers we supply for free?
  • fill out an on-line sequence order form including the declarations on Genetically Modified Organisms (GMO) and VAT.
  • send all of the above to the address on the bottom of the order form.

What concentrations of DNA and primers do I need to send?

We find that sequencing reactions are quite robust but we do need to know roughly what these values are. We aim for the concentrations of DNA to be 1ng/μl per 100bp for PCR products, 100ng/μl for plasmids and 3.2pmol/μl for oligos. Volumes supplied should be 6μl per sequencing reaction requested of plasmid or PCR product and 10μl of your oligos per reaction requested.

 

Do PCR products need to be cleaned before being sent for sequencing?

PCR templates for sequencing must have been cleaned up to remove unincorporated reactants before sending to us. In our hands all the commercial kits work OK. We will not accept liability for failed sequences when in our opinion the templates are poorly purified.

 

Will you make DNA template?

Yes. We have a standard protocol for extracting plasmids and for making PCRs. If you send us cultures for plasmid extraction then you must complete the declaration on GMO on the sequencing order form. If you fail to complete this then any material you send us will probably be destroyed. PCR primers must accompany PCR reaction requests. We make only one attempt at these using our standard conditions or conditions that you supply. If you decide that we need to work further on the PCR (titration) then further costs will be incurred. It is always best to contact us in advance of making template preparation requests.

 

Do you make oligos?

No. We are not an oligo supplier although we do have stocks of standard primers that we use at no charge to customers on their experiments. We do have a preferred oligo supplier and can get oligos synthesized quickly and cheaply for customers.

 

What are your standard sequencing primers?

M13F, M13R, T7F, T7R, T3, SP6, SK, KS and growing every day! The sequences are available on our web site.

Tell me about your registration to handle genetically modified organisms (GMO)?

We are registered with the Health and Safety Executive (HSE) for the safe handling of your GMO. This registration is only valid if the GMO declaration is completed by you when sending cultures. We are unable to accept GMO when this declaration is not completed and your materials will probably be destroyed.

How much does all this cost?

Not very much! Sequencing prices are falling all the time and we pass on these savings immediately to our customers. However, like all businesses we recognize the benefits of economies of scale. This means that the more sequencing you send (or commit to in any year) the lower your price will be.

So you're doing it on the cheap?

On the contrary. Our sequencing operation uses over £½ million worth of hardware, including the latest ABI 3730 DNA sequencing technology.

What experience have you got then?

Source BioScience LifeSciences has been around, in the form of HGMP, MRC Source BioScience LifeSciences and the Linkage Hotel at the HGMP-RC for almost 10 years. The sequencing team has more than 10 years experience of making reads. In the past 5 years members of our team have contributed over 2 million reads to the UK Mouse Sequencing Consortium and then the Fugu sequence finishing project.

So you only deal with large projects?

No. The minimum number of reads that we accept is 1.

What read lengths do you get?

Unlike some sequencing operations we don't make any promises on sequencing read length - we know that in isolation these promises are always wrong. On average we get about 750bp to 850bp of quality clipped sequence, although our reads are much longer at over 1kb.

 

But your competitors promise longer reads than that!

We can't make these promises and are surprised when others do. There are lots of reasons why sequencing reactions fail and most of them are nothing to do with us or you - that's biology. Repetitive elements and secondary structure account for short reads as will poorly purified templates. Of course a PCR product of 600bp will never give you a 650bp sequencing reaction. All of these things bring our averages down from the actual standard read length of a sequencing pass that is much higher.

 

What can you do about these biological sequencing fails?

We have a number of strategies to improve templates (Template Rescue Service, TRS) and to cover repetitive sequence or sequence with secondary structure (Secondary Structure Solution, SSS). We purchase some commercial reagents and have a few secret tricks for crossing these regions. Please contact us if you have a special requirement.

Do I pay for sequencing reaction fails?

Yes. We have controls in place such that we are confident that sequencing fails are generally the responsibility of the customer because of poorly prepared or purified templates, or bad oligos. You will always be sent failed read chromatograms for you to inspect them. However, when reads are sent to you, you can always ask for failed reads to be repeated and we are very happy to do so. Experience shows ~95% of failed reads fail again. In this case we will charge double for the fail if we repeat it. Obviously, if the failed read passes (5% of occasions) on repeat, we concede this to be our responsibility and you only pay once.

 

What is your turn-around time?

Between 1 and 2 days but this depends on the level of contract research requested and the number of reads. If you want to send us more than 90 reads at once we suggest you contact us in advance. Machine breakdown has caused turnaround time to be longer than this but we always keep a capillary sequencer in reserve.

 

How is data returned to me?

Most customers want their reads to be delivered by email attachment. For larger orders we can set up secure FTP sites or make CD-ROMs. Please contact us if you have any special requirement.

 

How do I know you won't make an error with my important samples?

We have laboratory information management systems (LIMS) in place. Your material is assigned a 96 well plate names and position when we receive it. From then on it is handled in this format and tracked with bar coding at every stage. We do not promise that mistakes cannot be made - we do promise that we have minimised these chances and that we will strive to always get you the best possible data. We will always be honest with you about your sequencing and your plates controls are always stored and are available for you to see. In the event of any dispute between customers and ourselves we will generally offer to make repeat samples for free.

 

I still have some questions...

OK, contact us now.