Genetic determination of Next Generation sequencing technology error rates within microsatellite sequences

Abstract

Previous research have shown that microsatellite sequences are underrepresented after massively parallel, Next Generation (NextGen), sequencing approaches compared to the known number detected in the human genome project. The goal of this project is to identify the accuracy and efficiency in reading repeat sequences in both sequencing technologies. To study this mono‐ di‐ and tetra‐nucleotide repeats were inserted in‐frame within the Herpes Simplex Virus1–thymidine kinase gene. Sanger sequencing and selective plating verified correct vector constructions. In addition, we specifically determined the mutation frequencies obtained during NextGen DNA library preparation of the plasmids. The starting background mutational frequencies(MFB) of all plasmids were determined by electroporation of E‐coli and selective plating. The NextGen library preparation steps were mimicked with each of the plasmids. DNA fragments were isolated by gel purification, amplified, and electroporated in E‐coli to determine the MFQ and MFQ+PCR. The degrees of technology‐introduced errors were calculated by comparing MF after amplification to background MF. From this we determined that all microsatellites were susceptible to technology‐introduced errors. The experimental design can be used to compare errors introduced during DNA library preparation. Funding MS Life Cycle NIH Grant GM087474.