Safe-SeqS

Safe-Sequencing System is a Unique Molecular Identifier (UMI) Approach to Detect Rare Variants

Safe-SeqS (or, more commonly, Safe-Seq) is an approach that uses unique molecular identifiers (UMIs) to detect rare variants (Kinde et al., 2011). In the time after the publication of the method in 2011, the use of UMIs has become ubiquitous, particularly in single-cell sequencing approaches, and the name of the method fell into disuse.

Safe-Seq assigns a UMI to each template molecule and amplifies each uniquely tagged template molecule to create UMI families. The abundance of each UMI can be used to distinguish between rare mutations and technical errors, as well as to correct for PCR amplification bias.

Advantages:

  • Distinguishes between rare mutations and technical errors
  • Detects 1 mutant template among 5000 to 1,000,000 wild-type templates (Kinde et al., 2013)
  • Corrects for PCR amplification bias

Disadvantages:

  • Can introduce spurious cross-hybridization
  • Complex protocol that requires a gel-purification step (Stahlberg et al., 2016)
  • Uses 2 or 4 PCR cycles for barcode addition and does not satisfy the basic principle of labeling each molecule with a single, unique barcode (Kukita et al., 2015)


Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Gregory M. T., Bertout J. A., Ericson N. G., et al. Targeted single molecule mutation detection with massively parallel sequencing. Nucleic Acids Res. 2016;44:e22

Wang K., Ma X., Zhang X., et al. Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis. Sci Rep. 2016;6:25310

Belic J., Koch M., Ulz P., et al. Rapid Identification of Plasma DNA Samples with Increased ctDNA Levels by a Modified FAST-SeqS Approach. Clin Chem. 2015;61:838-849

Do H. and Dobrovic A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem. 2015;61:64-71

Heitzer E., Ulz P. and Geigl J. B. Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem. 2015;61:112-123

Maslov A. Y., Quispe-Tintaya W., Gorbacheva T., White R. R. and Vijg J. High-throughput sequencing in mutation detection: A new generation of genotoxicity tests? Mutat Res. 2015;776:136-143

Patel K. M. and Tsui D. W. The translational potential of circulating tumour DNA in oncology. Clin Biochem. 2015;48:957-961



References:

Dal Molin M., Zhang M., de Wilde R. F., et al. Very Long-term Survival Following Resection for Pancreatic Cancer Is Not Explained by Commonly Mutated Genes: Results of Whole-Exome Sequencing Analysis. Clin Cancer Res. 2015;21:1944-1950

Eboreime J., Choi S. K., Yoon S. R., Arnheim N. and Calabrese P. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing. PLoS One. 2016;11:e0158340

Wang Y., Springer S., Mulvey C. L., et al. Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med. 2015;7:293ra104