NEXT-GENERATION SEQUENCING continued
Table 3 – SNP frequency of nucleotides determined from manual and automated performance
Figure 2 – Standard curve of the magnetic bead suspension prepared by manual suspension (n = 3).
Table 4 – Correlation of manual and automated libraries
Automated library versus positive control manual library (r2 0.997 0.995 0.995 0.993
)
0.994 0.995
libraries (Table 3). Manual and automated library correlations (r2 ) for both
the positive and negative controls (Table 4) demonstrate the system’s high reproducibility; this can be attributed to the uniform distribution of magnetic beads and accurate pipetting.
Figure 3 – Automated distribution of the magnetic bead suspension (automated mixing) to the target well of the multiple-well plate for nucleic extraction or purification (n = 3).
Table 2 – Purified amplified library concentrations for positive and negative controls from the manual and automated procedures within the acceptable limits
References 1. Grada, A. and Weinbrecht, K. Next-generation sequencing: methodol-
ogy and application. J. Investigative Dermatology 2013, 133, 248–56.
2. Schneeberger, K. Using next-generation sequencing to isolate mutant genes from forward genetic screens. Nature Rev. Genetics 2014, 15, 662–76.
3. Zhang, L.; Chen, L. et al. Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing. Oncologist 2014, 19(4), 336–43.
4. van Wezel, E.M.; Zwijnenburg, D. et al. Whole-genome sequencing identifies patient-specific DNA minimal residual disease markers in neuroblastoma. J. Molec. Diagnostics 2015, 17(1), 43–52.
5. Gill, S.; Gill, R. et al. Automated bead mixer for VERSA Mini Nucleic Acid Purification/PCR setup workstation. Laboratory Focus July 2011, 8–10.
6. Rodriguez, R. and Miller, K.M. Unravelling the genomic targets of small molecules using high-throughput sequencing. Nature Rev. Genetics 2014, 15, 783–96.
7. Zentner, G.E. and Henikoff, S. High-resolution digital profiling of the epigenome. Nature Rev. Genetics 2014, 15, 814–27.
AMERICAN LABORATORY • 8 • MARCH 2015
Automated library versus negative control manual library (r2 0.998 0.998 0.999 0.982
)
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