From GenoSeq

Understanding your Results

A Brief Introduction:

Examining the quality of the sequencing reaction in terms of signal strength is the single most important thing a novice sequencer can do. This is easily done by looking at the "raw data view" for your sequencing trace. Once you have established that there is a strong signal and yet the result is still poor only then can you begin to figure out what the problem might be.

On the y-axis low relative peak amplitudes indicate detection ability is approaching its lower limit. When the signal is low the quality of the sequence is poor and read length short. A low signal indicates a low amount of sequencing product is present. In most cases this is due to inadequate amounts of template in the sequencing reaction. A wide range of template DNA concentrations is tolerated by the sequencers we use and it is better to err on the side of more and not less.

Signal above 28,000 relative peak amplitude is approaching the maximum limit indicating too much sequencing product is present. This situation rarely occurs, however, if it does, the reaction can be diluted and run again. This must be done soon after the initial run.

Strong signal

Weak signal

Overloaded signal

Examples of Typical Sequencing Issues:

Dye blobs

Dye blobs occur around 79 bps into the sequence. They are the result of having an unused excess of BigDye in the sequencing reaction. If you need to read sequence in this region there are two simple ways to reduce dye blobs: Use less BigDye in your reactions (1 µl per 10 µl reaction is more than enough). Increase the amount of template DNA in your reaction so that the BigDye is used up in the sequencing reaction. A combination of these two methods will usually get rid of the dye blob. Above is a picture of a dye blob occurring in its usual position of approximately 79bp into the sequence there may sometimes be a smaller blob around 115 bps. Dye blobs are a regular feature in the core's Full Service results and otherwise good quality results are not re-done because of them. If it is essential for you for read through the region obscured by a dye blob we will ask you to provide the template at a several-fold higher concentration.

Double Sequence due to Multiple Clones

Notice after the EcoR1 site [GAATTC] the sequence is double. This happens when more than one clone is present for use as template. The vector region is the same for both clones and hence sequences fine, however, upon reaching the insert region it becomes apparent that a mixed template is present. Always be careful to pick one individual clone for your DNA preps

Double Sequence due to Homopolymers or Heterozygous Template

This phenomenon happens after stretches of homopolymers or repeats. It is believed to be due to DNA strands re-annealing off-center in the repetitive region during sequencing. Try sequencing from the complimentary strand if possible. If your template is a PCR product from diploid genomic DNA this shift may indicate a possible insertion or deletion in one of the alleles.

Premature Signal Loss

Due to secondary structures such as hairpins in the template that block the polymerase’s progress. Regions of G and C homonucleotides are also particularly problematic. Check the “raw data” view to make sure this pattern is simply not due to a just a low yield reaction where the signal started out low from the beginning. In this situation, changing the sequencing reaction's thermocycler temperatures to higher annealing and denaturing temperatures (Kieleczawa J., 2006) along with doubling the size of the reaction to 20 µl can be the most helpful.

Fundamentals of Sequencing of Difficult Templates. (2006) Kieleczawa J., Journal of Biomolecular Techniques,17:207-217. [1]