Vibration as a pitfall in pyrosequencing analyses

Abstract

Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful validation and establishment of several pyrosequencing assays using a PyroMark® Q48 Autoprep instrument (Qiagen, Hilden, Germany), we decided to expand the method further purchasing a second instrument. But after initializing this second instrument side by side with the first, the majority of analyses failed (97 samples of 133 samples (73%)). The number of error messages increased rapidly and the average RFU values decreased. After purchasing two anti-vibration weighing tables for the PyroMark® instruments and repeating the analyses under the same conditions and with identical samples the results improved considerably, 115 samples of 130 samples (88%) showed successful and reproducible results. These findings demonstrate the impact of vibrations and percussions on PyroMark® Q48 Autoprep performance and the reliability of methylation analyses.