Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D; community with critical information on the tools, technologies, and trends that drive the biotech industry.
Issue link: http://gen.epubxp.com/i/350894
20 | AUGUST 2014 | GENengnews.com | Genetic Engineering & Biotechnology News Single-Cell Approach to Personalized Medicine OMICS Yan Zhang, Ph.D., is market analyst and Joyce Peng, Ph.D. (joyce.peng@bgiamericas .com), is marketing director at BGI Tech. Website: www.bgitechsolutions.com. Yan Zhang, Ph.D., and Joyce Peng, Ph.D. As one of the most prevalent causes of death across the globe, the burden of cancer is still sharply increasing, predominantly due to the aging population. As such, the current focus of many researchers is on how to accurate- ly diagnose and treat various cancer types. However, human cancers usually carry sev- eral different genomic variations, such as copy number variation and point mutations, which essentially lead to tumor heterogene- ity. These tumors therefore display different cellular morphology, gene expression, me- tabolism, motility, proliferation, and meta- static potential. This phenomenon occurs both within individual tumors and between different tumors in the body. This inherent variation of cancer cells causes signifcant issues in the development of targeted therapies. Drug development has previously focused on the genomic dif- ferences between complex mixtures of cells, employing techniques that may obscure the heterogeneity of single cells, leading to the development of less effcient treatments. Single-cell genomics can facilitate the eluci- dation of cell lineage relationships. Individual cells can be isolated using micromanipulation with a mouth pipette or by serial dilution and the genome sequenced using next-generation sequencing technologies. Through a com- parison of the genomes of individual cells, re- searchers can determine the mutation profle infuencing a change in cellular morphology in the subsequent generation of cells. The major applications of this technique include profling scarce clinical samples (i.e., circulat- ing tumor cells), pre-implantation genetic di- agnosis, measuring intra-tumor heterogeneity, guiding chemotherapy, and cancer cell evolu- tion analysis during tumor progression. Techniques—Whole- Genome Amplifcation There are several different techniques that can be implemented to amplify the genomes of single cells for sequencing. There are how- ever pros and cons associated with each of these, which will impact the quality of the sequence data: PCR-Based Methods A staple of the molecular biology labora- tory, PCR is an accessible and reliable ampli- fcation technology. • Degenerate oligonucleotide-primed PCR (DOP-PCR) uses semi-degenerate oligos (where a section of the oligo uses degenerate bases—essentially a mixed base, which is ide- al for amplifying an unknown target) and an increasing annealing temperature. • Primer extension pre-amplification PCR employs a pre-amplifcation step to add binding sites to the fragmented genome, al- lowing whole-genome analysis. This proto- col uses random primers and a low anneal- ing temperature. However, the use of Taq polymerase limits fragment length to 3 kb and can introduce sequence errors. Both the primer extension and degener- ate oligo methods have been found to exhibit amplifcation bias, where a sequence is over- represented in the amplifed DNA due to pref- erential binding of primers to specifc regions. • Multiple annealing and looping based amplifcation cycles (MALBAC) is a newly developed PCR-based method of whole- genome analysis. It uses quasi-linear ampli- fcation to provide uniform resulting data, while generating low rates of false positives and negatives. The genome coverage at the single-cell level is less uniform than when sequencing in bulk; this approach is unable to detect approximately one-third of SNPs compared to bulk sequencing. Non-PCR-Based Methods • Multiple displacement amplifcation (MDA) can rapidly amplify minute quanti- ties of DNA. Hexamer primers are annealed to template DNA, and the synthesis is per- formed using a high-fdelity polymerase at a constant temperature. Compared to con- ventional PCR techniques, MDA generates larger products (the average product length is >10 kb) with lower error rates, which are optimal for the detection of copy number variation and structural variation. Advancing the possibilities of single-cell sequencing in human research, BGI Tech (a Improving Cancer Detection and Therapy Figure 2. Key gene identifcation of the patient. The driver gene prediction analysis of the 18 candidate genes is indicated as a Q score. The vertical axis is the Q score, and the circle diameter indicates the cell mutation frequency. Figure 1. Method of sequencing samples from a JAK2-negative myeloproliferative neoplasm patient. Tutorial Editor-in-Chief: Hod Lipson, PhD The frst peer-reviewed Journal to explore emerging challenges and opportunities in additive manufacturing and related technologies Sign up for TOC Alerts www.liebertpub.com/3DP To receive a complimentary copy of this journal, please email smohin@liebertpub.com with the subject line: 3DP Journal