We occasionally get asked if the PeakTrace Basecaller™ ever been independently validated? The answer is no one has ever published an independent validation of any Sanger DNA sequencing basecaller. This includes the widely used KB Basecaller™.

The most complete published study on basecaller validation is Ewing and Green’s 1998 paper on the validity of phred [1]. This study was performed by the same team that developed phred [2] so while very though, it is far from an independent study.

The KB Basecaller™ has no published studies on its validation beyond a single poster at Advances in Genome Biology and Technology (AGBT) in 2004 by the team that developed KB [3]. While this poster showed that KB gave more Q20+ bases than phred, these extra Q20+ bases didn’t seem to help much when the data was used (i.e. in assemblies – see Figure 5 on this poster).

The ABI 3500 DX model has been approved for clinical testing by the FDA [4], however, the validation of KB done for this approval was performed by ABI and is limited to the ABI 3500 DX model when using POP6™, BigDye™ v1.1, the 3500 Dx Series Data Collection Software 1.1, and one of two run conditions [5]. If you are running any other sequencing instrument, polymer, BigDye, or run condition then KB has not gone through the FDA validation process. You can’t assume that KB will give valid results under your run conditions just because it has been validated for a particular instrument, consumable set, and run conditions.

It is totally understandable why ABI (ThermoFisher these days) has only undertaken the FDA approval process for the ABI 3500 DX and only under a very limited range of conditions. The FDA process takes years, costs millions of dollars, and needs to be done separately for each instrument, polymer, terminator chemistry, capillary length, and run module. Even for a company as large as ThermoFisher it does not make sense to go through the FDA process for every sequencer it has ever sold.

The closest to an independent study of the KB Basecaller we have been able to find was a paper published by Hyman et. al. in 2010 [4]. They did not perform a full validation (as was done by Ewing and Green for phred), but they did find that KB gave more Q20+ bases than phred. They also found that these extra Q20+ bases did not help them in their application of identifying bacterial species from the sequence data [4].

In defence of the KB Basecaller our own validation studies shows that KB is basically fine except for a few problems in the Q20 to Q30 range where it tends to over predict the actual quality [7]. For almost all non-clinical applications this is not an major issue, but it may explain why there has been little or no benefit seen from the greater Q20+ bases that KB provides over phred.

The bottom line is KB (like phred) has been validated by the people who wrote it, but no one has published an independent validation of either of these basecallers except ironically us [7]. If you demand that a basecaller be independently validated before you will use it then the only independent study you can turn to is ours of KB (we have also validated phred, but this has not been made public). If you trust us to validate KB, then you can trust us to validate PeakTrace too.

So where does this leave the validation of the PeakTrace Basecaller? Nucleics has extensively validated PeakTrace [7] in exactly the same manner as was done by the developers of the phred [1] and KB basecallers [3]. PeakTrace is currently being used in 93 facilities around the world to basecall tens of millions of traces per year. Our customers would not pay the extra cost for using PeakTrace – after all they get the KB Basecaller for free with the instrument – if they did not believe it provided real and significant improvement.

Of course the best validation is always your own validation. For almost all applications it does not matter if, for example, a predicted Q31 base has a true Q31 error rate (or if it is actually Q29 or Q33), what matters is the number of actual errors, the usable read length, and that bad bases are not called as good bases. We have yet to see an application (even clinical) where it matters if a base is given a Q score more accurate than to the nearest Q10.

Luckily it is very easy to do a quick, do-it-yourself validation of PeakTrace. Just grab a handful of good traces of known sequence, run them through the free online PeakTrace service, then BLAST both the KB and PeakTrace sequence data to comparing the errors and aligned bases found with each bascaller. This will quickly tell you which basecaller is best for your data. If you follow this process we are sure you will be impressed with how PeakTrace performs.

References

1. Ewing B, & Green P. (1998). Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8:186-194.
2. Ewing B, Hillier L, Wendl MC, Green P. (1998). Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175-185.
3. Longer Reads and More Robust Assemblies with the KB Basecaller (2004)
4. http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/SubstantiallyEquivalent510kDeviceInformation/UCM339686.pdf
5. https://tools.thermofisher.com/content/sfs/brochures/3500-dx-series-genetic-analyzer-cs2.pdf
6. Hyman RW, Jiang H, Fukushima M, Davis RW. (2010). A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry. BMC Research Notes. 3:257.
7. Tillett D. (2010). Validation of the PeakTrace Basecaller.