1. In Silico Proficiency Testing for Clinical Next-Generation Sequencing
Eric J. Duncavage, Haley J. Abel, John D. Pfeifer 
The Journal of Molecular Diagnostics 
Volume 19, Issue 1, Pages (January 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Generation of in silico proficiency testing (PT) sequence files. Two basic methods exist for the generation of in silico PT. A: In the simulated or de novo method, a mutation (red text) is introduced into the gene of interest by altering the corresponding position in the reference sequence. Reads are then simulated from the mutated reference sequence using estimated error rate and coverage models. Although simple to implement, such approaches may not accurately simulate the true error rate and may produce coverage distributions that do not accurately reflect real clinical data. B: Mutations can also be simulated by introducing variants into actual sequence reads from clinical cases. Using this approach, software tools modify the individual reads from aligned data to include the mutation and associated base quality score in a given fraction of reads to simulate the wanted variant allele frequencies. Alignment data are then stripped from the mutated reads, allowing for regeneration of raw sequencing files. Although more technically complex, the major advantage of this method is that it accurately simulates expected coverages and error profiles (maroon text).
The Journal of Molecular Diagnostics  , 35-42DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions