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Sample Requirements

Sample Requirements & Sample Analysis

Preparation of samples

Volume

We need 5µl per reaction of both your primer and DNA at the concentrations specified below:

Plasmid DNA

100ng/µl

PCR product 

1ng/µl per 100bp

Bacterial culture  (Bugs2Bases service)

150µl of overnight culture provided in a 96-well plate 

Primers  

3.2pmol/µl

DNA concentration needed

  • Plasmid: We require a concentration of 100ng/ul for plasmid samples.
  • Purified PCR samples: The concentration of DNA required is dependent on the length of the sample sent. A concentration of 1ng/ul per 100 base pairs is required for successful sequencing.  

Primers

We require your primers to be sent at 3.2pmol/ul and require 5µl per reaction. You can choose to send your own primers, use our stock primers or you can order custom primers that will be delivered to the sequencing laboratory.

We recommend the following when designing your primers:

  • Choose a suitable primer length (~18 - 23 base pairs)
  • Design your primers at least 50 bases upstream of your area of interest
  • Design primers with a tm between 55°C and 60°C
  • Ensure your primers have a GC content between 40 and 60%. 

Quality of samples

All samples must be of a good quality, free from contaminants such as salts, other contaminating DNA, unincorporated dinucleotides (in PCR reactions) and other reagents that would interfere with the reaction.

Prior to submission, we advise running your samples on agarose gels to ensure only one clearly defined band is visible on the gel (i.e. that your DNA is not contaminated with other DNA/it has not degraded).

It is important that you are aware of any motifs or secondary structures that might impede high quality sequencing results. If you know that this is most likely going to be a problem we recommend you use our secondary structure resolution option when ordering.

Receiving results

In order to receive your results as soon as possible it is advisable that you choose our free SpeedREAD™ service. SpeedREAD™ is an automatic service which dispatches your results as soon as the sequencer has completed the analysis. If you choose this option you will receive an automatic email with a link to your results. This will be followed up with an email from our sequencing team the following working day. This email will include the results of their quality checks on your samples, and should samples fail, the sequencing team will advise you as to reasons why they failed and potential solutions.

What good sequencing traces look like

Below (Figure 1 and Figure 2) are examples of good sequencing traces of a high quality. Figure 1 displays a plasmid sample which has produced a good sequencing trace.  Figure 2 is a sequencing trace from a PCR sample. PCR samples produce peaks of equal height the whole way through the sequence. This raw data gives us information on the length of the read, and the height of the peaks which in turn determines whether or not the peaks are within acceptable heights.  Figure 3 is an electropherogram for a good quality sequencing trace. Note it has well defined peak resolution, uniform peak spacing and has a weak background noise in comparison to the sample.  

 Plasmid sample

Figure 1 - Raw data for a plasmid sample which gives a good quality sampling trace

PCR Sample

Figure 2 - Raw data for a PCR sample which gives a good quality sequencing trace

Raw PCR Sample

Figure 3 - Electropherogram for a sample which gives a good quality sequencing trace

Most common reasons why sequencing reactions fail

Samples too concentrated

Figure 4 is the raw data from a PCR product that was too concentrated. As shown in the figure, the results reach above the highest detectable levels on the graph. Figure 5 is the corresponding electropherogram for this sample. Note the peaks are poorly defined, overlap and are not uniformly spaced. The sample produced what would be known as a mixed trace, due to the poorly resolved base calling in the electropherogram. Figure 6 shows a plasmid sample which is too concentrated, the sequencing trace is very concentrated at the beginning and it decreases rapidly producing a shorter than expected sequencing trace.

Figure 4 - Raw data for a PCR sample which is too concentrated

PCR Reaction

Figure 5 - Electropherogram for PCR reaction which is too concentrated

Figure 6 - Raw data for a plasmid sample which is too concentrated

Low sequencing trace

Figure 7 and Figure 9 display failed sequencing traces. On the raw data (Figure 7) the scale is low and there is no obvious increase in fluorescence. Figure 8 shows the electropherogram for data from Figure 7. No bases are called and they are all assigned with the letter N. In Figure 9 the sample has failed due to a low concentration of DNA in the sample, (there is some binding, but at a low level of fluorescence). Figure 10 is the electropherogram for Figure 9, which shows bases are not being called with a high quality of confidence.

Raw data

Figure 7 - Raw data for a sample that has not produced any results

Electropherogram Sample Sequencing

Figure 8 - Electropherogram for sample which has not produced a sequencing trace

PCR Sample

Figure 9 - PCR sample which has failed due to a low concentration of DNA in the sample

PCR

Figure 10 - Electropherogram for PCR sample which has produced a low fluorescence

Table 1: Potential causes for samples producing a poor sequencing trace

Probable cause

Solutions

Insufficient DNA concentration

Increase DNA concentration

Insufficient primer binding

Redesign primer

No primer binding site present

Redesign primer or use different primer

Degraded DNA

Re-extract DNA template or clean up template

Degraded primer

Make up new primer stock


Secondary structures

Secondary structures are unexpected early terminations of your sequence (Figure 11). These can be due to your template having a high number of GC rich areas, which have a tendency to cause the DNA to loop and form a hairpin bend. The polymerase cannot continue the reaction and therefore the sequence terminates early.

Secondary structure

Figure 11 - Figure of a secondary structure. The arrow shows where signal is lost due to the secondary structure

Table 2: Potential causes for early termination of a sequencing trace 

Probable cause

Solutions

Secondary structure present in your DNA

  • Choose secondary structure resolution option when ordering
  • Redesign primers to avoid the formation of the secondary structure


Mixed traces

Several different mixed traces can be produced dependent on the cause. If the trace has produced a mixed trace continuously through the electropherogram (Figure 12), it is most likely due to either your sample containing multiple DNA templates or your primer is binding to more than one region of your DNA.

Single nucleotide polymorphisms (SNP) may also cause what would appear as a mixed trace at one particular base, with no affect the rest of the sequencing trace (Figure 13). An insertion or deletion in the DNA however, will appear as normal sequence up until the insertion or deletion occurs, followed by a mixed trace (due to the multiple sequences are now being produced). A mixed trace may also occur due to enzyme slippage, this can occur after a homopolymeric region (Figure 14), which causes the growing strand to unpair correctly with the template DNA during polymerization.

Mixed trace Electropherogram

Figure 12 - Electropherogram showing a mixed trace

SNP's

Figure 13 - Electropherogram showing SNPs

Figure 14 - Electropherogram showing enzyme slippage following a homopolymeric region

Table 3: Potential causes of mixed sequencing traces

Probable cause

Solutions

Mixed templates present in sample

Re-isolate DNA

Frame shift mutation

Use different primer after the mutation or sequence the complimentary strand

Multiple primer binding sites

Ensure the primer will only bind at one spot

Enzyme slippage due to a homopolymeric region

Sequence the complimentary strand


Dye Blobs

Unincorporated dye-terminators (commonly called dye blobs) appear at positions 70 to 80bp, and again at approximately 100bp. The chromatogram below shows unincorporated dye-terminators superimposed over, and partially obscuring the real peaks.

Dye blobs are caused by an imbalance of primer:BigDye:template. We use proprietary clean-up plates to remove dye blobs but in extreme cases of imbalance they can still remain. We have optimised primer: BigDye concentrations for specified template concentrations. Therefore it is important that you ensure to send the correct template concentration should you find that dye blobs are a problem for you.

 

For further information and prices please contact us or call +44 (0)115 973 9012

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