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Genetic Engineering & Biotechnology News | GENengnews.com | AUGUST 2014 | 21 OMICS subsidiary of BGI) has developed an innova- tive end-to-end solution for genomics and transcriptomics analysis at the single-cell level. Based on the fndings mentioned above, MDA has been further enhanced and incor- porated into BGI's whole-genome amplifca- tion protocol, within the single-cell sequenc- ing service. Following whole-genome ampli- fcation, quality control protocols employ eight housekeeping genes as internal indica- tors, thus identifying sequence-ready samples in a cost-effective manner, without affecting the genes of interest. Combining whole-genome amplifcation with next-generation sequencing, BGI Tech is able to obtain single-cell sequence data for disease research and the development of per- sonalized medicine. The methodology can be applied to discover genetic information with- in single cells, allowing for the identifcation of the mutations associated with the devel- opment of cancerous cells, and consequently those that play a more causal role in disease progression and metastasis. Case Study Tumor heterogeneity presents a signifcant challenge when attempting to determine clonal evolution and the identifcation of the genes underlying cancer progression. Here, we ana- lyze tumor evolution and the intratumoral het- erogeneity of myeloproliferative neoplasm (1) and clear cell renal cell carcinoma (ccRCC) (2). Both cases confrm high heterogeneity within cancer cell populations, with several novel can- didate mutations identifed. Thus, single-cell sequencing is proven to generate a fresh wave of biological insights into cancer research. Case 1—Myeloproliferative neoplasm Single-cell sequencing of a sample from a patient with a myeloproliferative neoplasm was performed to reconstruct tumor ancestries and identify the mutations driving this cancer. 1. High-throughput whole-genome single-cell sequencing was conducted on two single cells from a lymphoblastoid cell line. Whole-genome recovery, amplifcation uniformity, sensitivity, and specifcity were evaluated. 2. Whole-exome single-cell sequencing was performed on 90 single cells from the patient. The sequencing data from 58 cells passed the quality control criteria, and these data indicated that this neoplasm represent- ed a monoclonal evolution (Figure 1). Fifteen genes were found to be likely to contain protein-damaging mutations (in- cluding protein truncation). Eight genes were subsequently identifed, which had a signif- cantly higher prevalence of protein-function- alternative somatic mutations (Figure 2). A single-cell sequencing method was therefore established, which provides detailed analysis of a variety of tumor types, including those with high genetic variation between patients. Case 2 - Clear cell renal cell carcinoma ccRCC is the most common kidney can- cer, but the patients share very few common mutations. To investigate the intratumoral heterogeneity of ccRCC, single-cell exome se- quencing was conducted on a ccRCC tumor and its adjacent kidney tissue, enabling the generation of a detailed intratumoral genetic landscape at the single-cell level (Figure 3). Quantitative population genetic analysis indicated that the tumor did not contain any signifcant clonal subpopulations and that mutations that had different allele frequen- cies within the population also had different mutation spectrums. Conclusion Research into the development and pro- gression of cancer is a complex task. Con- ventional strategies for cancer research are often labor intensive and limited by through- put, making them ineffcient. The use of next-generation sequencing for genome-wide variant analysis therefore presents a signif- cant development within this feld. Since can- cer is principally a disease of accumulated genomic alterations, whole-genome analysis will help to devise novel personalized strate- gies for cancer diagnosis and therapy. As demonstrated here, single-cell se- quencing has been proven as a valuable tool within human disease research. However, the development and fne-tuning of these methods is still in progress. Single-cell isola- tion is crucial for sequencing effciency, and BGI Tech is currently in the process of testing new automated cell-isolation technologies with the aim of enabling accurate sequencing of single cells to consequently develop even more effective cellular targeted therapies. It is also expanding its R&D; pipeline to enable the expansion of single-cell applications to cover stem cells and the human brain. 10³ 10⁶ OVA OVA + alhydrogel® OVA + IFA OVA + ODN1826 OVA + c-di-GMP OVA specifc antibodies in serum of immunized mice were evaluated 30 days post subcutaneous injection with the OVA antigen alone or with diferent InvivoGen adjuvants. Total m1gG anti-OVA levels Tailor the adaptive immune response. Pick and mix to master the bias. Intensive research focuses on optimizing the use of vaccine adjuvants to not only enhance but orientate specifc Th1 or Th2 adaptive immune responses to antigens. Boost your adjuvant research by choosing from the number one adjuvant collection. InvivoGen provides an extensive range of adjuvants including the approved alum and oil emulsions, and the promising pattern recognition receptor (PRR) ligands. Shape Immunity VACCINE ADJUVANTS www.invivogen.com/vaccine-adjuvants VacciGrade ™ guarantees purity and sterility Largest choice of vaccine adjuvants Create your own adjuvant mix Design your Adjuvants Figure 3. Research strategy for single-cell exome sequencing of a ccRCC tumor and its adjacent kidney tissue. Tutorial