Safe-SeqS

Safe-SeqS

Safe-sequencing system (Safe-SeqS), or more commonly Safe-Seq, is the name given to a unique molecular identifier (UMI) approach to detect rare variants. Since the publication of the method in 2011 the use UMIs have become ubiquitous, particularly in single cell sequencing approaches, but the name of the method fell into disuse.

Safe-Seq assigns a unique identifier (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 and it can also be used to correct for PCR amplification bias.

Pros:

• Distinguish between rare mutations and technical errors
• Detect 1 mutant template among 5000 to 1,000,000 wild-type templates¹
• Correct for PCR amplification bias

Cons:

• Can introduce spurious cross-hybridization
• Complex protocol that requires a gel-purification step²
• Uses two or four PCR cycles for barcode addition and does not satisfy the basic principle of labelling each molecule with a single unique barcode³

• Safe-SeqS: Kinde I., Wu J., Papadopoulos N., Kinzler K. W. and Vogelstein B. (2011) Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A 108: 9530-9535
• 1. Kinde I., Bettegowda C., Wang Y., Wu J., Agrawal N., et al. (2013) Evaluation of DNA from the Papanicolaou test to detect ovarian and endometrial cancers. Sci Transl Med 5: 167ra164
• 2. Stahlberg A., Krzyzanowski P. M., Jackson J. B., Egyud M., Stein L., et al. (2016) Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing. Nucleic Acids Res 44: e105
• 3. Kukita Y., Matoba R., Uchida J., Hamakawa T., Doki Y., et al. (2015) High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients. DNA Res 22: 269-277

1. Dal Molin M., Zhang M., de Wilde R. F., Ottenhof N. A., Rezaee N., et al. (2015) 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 21: 1944-1950
2. Eboreime J., Choi S. K., Yoon S. R., Arnheim N. and Calabrese P. (2016) Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing. PLoS One 11: e0158340
3. Wang Y., Springer S., Mulvey C. L., Silliman N., Schaefer J., et al. (2015) Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med 7: 293ra104