What is base calling?

Base calling is the process of assigning bases to chromatogram peaks. DNA Sequence Assembler has its own embedded base caller.

Conventional base callers

One of the oldest base calling program is Phred. The Phred base caller determines a sequence of base-calls from the processed trace in a four-phase procedure. In the first phase, idealized peak locations (predicted peaks) are determined; the idea is to use the fact that fragments are locally relatively evenly spaced, on average, in most regions of the gel, to determine the correct number of bases and their idealized evenly spaced locations in regions where the peaks are not well resolved, noisy, or displaced (as in compressions). In the second phase, observed peaks are identified in the trace. In the third phase, observed peaks are matched to the predicted peak locations, omitting some peaks and splitting others; as each observed peak comes from a specific array and is thus associated with 1 of the 4 bases, the ordered list of matched observed peaks determines a base sequence for the trace. In the final phase, the uncalled (i.e., unmatched) observed peaks are checked for any peak that appears to represent a base but could not be assigned to a predicted peak in the third phase, and, if found, the corresponding base is inserted into the read sequence. The entire procedure is rapid and take less than half a second per trace.

However, Phred was developed in '90. The algorithm used starts showing its age and its accuracy is not sufficient for modern times.

DNA Baser's embedded base calling

Starting with version 4, DNA Sequence Assembler implements a state-of-the-art base calling algorithm that is superior to any classic base caller. The algorithm is used to reassert base confidence score (also called QV or base trustfulness) and to detect mutations.

At the moment, DNA Baser Assembler is the first and only sequence assembler on the market that offers an embedded base caller!

DNA Baser Assembler uses the basecaller module to:

• Re-call undetermined based (N bases)
• Computing (or re-computing) the base QV (confidence score) in chromatograms
• Correct contig's ambiguities
• Detect double peak mutations in chromatograms

Recalling undetermined (N) bases

Some simplistic base callers will not be able to correctly resolve all peaks (call all bases). Especially in at the ends of the chromatogram where the quality of the electropherogram is poor. This will result in a sequence that contains undetermined (N) bases.

DNA Baser call pass all these unresolved bases through its accurate base caller and assign the correct base to them.

Example:

Below we can see bases in a chromatogram called with a 3rd party base caller:

And here is the same chromatogram processed by DNA Baser's base caller:

How to activate it?

In order to activate this feature, the user has to check the 'Recall N peaks' box.

Note: this is a one-time operation. Once activate it will remain active for all future projects.

Computing base's quality values (QV)

The user can choose if he wants to keep and use the precalculated quality values in a chromatogram (computed with his own basecaller) of if DNA Baser will recompute those values. In case the chromatogram contains no QV, DNA Baser will compute them anyway.

Example:

In the image below we can see how a 3rd party base caller assigned QV to the bases. All quality values are in the 90-95 range. No matter if the peak is high or low, if it overlaps with other peaks or not, the value is almost constant. Running the chromatogram through our base caller we obtain more realistic values, in the 0-71 range.

How to do activate it?

In order to activate this feature the user has to check the 'Use internal algorithm to calculate base quality' box.

Note: this is a one-time operation. Once activate it will remain active for all future projects.