Troubleshooting

This page discusses topics related to troubleshooting sequencing reactions. Sometimes, even experienced molecular biologists tend to forget these issues because nowadays most sequencing is outsourced to internal core facilities or private companies. Our experience confirms that keeping an eye on them can easily make the difference between a great result and a failed sequencing reaction. We hope that refreshing these concepts will help you to avoid any sequencing pitfalls and enable you to optimise your projects.

Sequencing versus PCR

The following table summarises the main differences between the sequencing reaction and PCR. Keeping these in mind can sometimes help to understand unexpected suboptimal sequencing results.

Feature:	Sequencing:	PCR:
Elongation	Unidirectional (1 primer)	Bidirectional (2 primers)
Amplification	Linear	Exponential
Primers	Use exact matches	Degenerated primers OK
Contamination	Relatively tolerant	Very sensitive

Comments:
Since the cycle sequencing reaction is unidirectional the quality of primer binding is more crucial than in the case of PCR. Unless the concentration of the contaminant is high, contamination is not an issue in sequencing since the effect is proportional to the concentration as opposed to PCR where it is exponential. The best sequencing results can be achieved if you follow these hints:

1. When sequencing PCR products you can expect optimal results if an internal primer is used as opposed to a primer of the PCR primer pair, because the ends of the product could be less accessible to the polymerase. If you do not have such a primer we suggest you first try with the primers of the PCR pair and in case of problems design an internal one.
   
   
2. Sequencing with degenerated primers can create problems and often requires special optimisations. Try to avoid sequencing with degenerated primers, unless you absolutely have to and please inform us if you do so.

Dye Terminator Chemistry

Most sequencing is carried out with labelled ddNTP terminators known as Big Dyes (by Applied Biosystems) or ET terminators (by Amersham Biosciences). The rate of incorporation during the cycle sequencing reaction differs among the four dyes due to their different molecular structures. In addition to primer issues, this is one of the reasons why forward and reverse sequencing of the same template can yield different results in terms of quality.

Sequencing Polymerase

During cycle sequencing the elongation step is performed by a modified version of a DNA polymerase (also know as Sequenase). As in the case of PCR the polymerase is subject to slippage that can result in additional background when sequencing through repeats or poly-N stretches. After such a poly-N stretch the sequence can be difficult or even impossible to read.

Secondary Structures

Hairpins and other secondary structures can create big challenges to sequencing teams because they make these regions less accessible to the sequencing polymerase.This results in crashes that can range from slight signal reductions to complete sequence stops. Templates that build strong secondary structures or even dimers because of their peculiar base composition can be extremely hard to sequence and usually require some very specific optimisation steps.

Sequencing Pipeline Issues

Although we take great care to deliver excellent data quality we cannot fully exclude the occasional technical issue. In such cases we obviously repeat the analysis for free. If necessary, we will ask you to send some more template DNA.

Information Needed for Troubleshooting

Complexity of troubleshooting can range anywhere from easy to extremely difficult. In the case of sample failures we cannot readily explain, we ask customers to provide us with some of the information listed below. Since it is normally not necessary to know all the details about the failed sample we ask for the relevant information on a 'case by case' basis. At Synergene we believe that good communication is essential for successful troubleshooting. Therefore, we always encourage customers to contact us to discuss or even plan their own sequencing projects with us.

Sample Information

It is often said that sample quality is the most crucial factor affecting the outcome of a sequencing reaction, which is true but not very accurate. Many parameters make up the so called 'sample quality'. Here is some sample information we might ask for:

DNA concentration
Purification protocols used
Vector information such as type, map, and sequence
Bacterial strain information
Primer sequences (if not standard)
Primer melting temperature (Tm)
Origin of sample and extraction method
Cloning history
Gel images

Template Sequence Information

As described above, the base composition of the template can influence the sequencing reaction. The following information might be useful for sequencing optimisation:

GC and AT percentage
Secondary structure such as hairpins and loops
Target sequence (if known)
Presence of repetitive elements such as LTRs, microsatellites, telomers, or regulatory regions
Presence of His-tags or poly-N stretches