The ICBR and UFHCC have partnered in the acquisition of new cutting-edge technology with the capability of analyzing gene structure and gene activity at the single cell level. The Chromium instrument developed by California-based 10X Genomics inc. provides two unique key technologies to the UF research community. In one application, the instrument uses synthetic gel beads to partition thousands of separate enzymatic reactions necessary for downstream DNA sequencing. In another application, short DNA sequencing reads of a few hundred bases can be linked together in correct order to generate longer DNA sequences representing very large contiguous segments of chromosomes. This facilitates the assignment of separate single base variants and mutations that may be separated by significant distance to either of the two normal copies of a gene. In the Chromium System this is accomplished by packaging very large DNA fragments into small reactions vesicles.
Each vesicle contains a unique “bar code”, such that all of the sequencing reads (which are much shorter than the original DNA fragment) derived from that very large DNA fragment can be linked back to the original fragment. This allows for assignment of particular single base variants to either of the two existing copies in the genome. [Current methodology only identifies the base variant but does not assign the variant to gene copy 1 or gene copy 2]. In addition, other types of structural variants, such as deletions or insertions, can also be assigned to the appropriate gene copy. Thus, separate mutations that occur in the same copy of the gene can be identified. In addition, creating very large regions of linked DNA sequencing reads facilitates the assembly of whole genomes – essentially putting all the DNA base pairs from a single genome into the correct order.
A second analysis feature of this instrument is that single cells isolated from a complex cell mixture (like tumor tissue, for example) can be packaged into the gel beads. The instrument can analyze from 1000 to 10,000 cells per experiment. RNA molecules present in each encapsulated single cell are converted into DNA that is tagged with a short DNA sequence tag that serves as an identifier that is unique to that single cell. DNA from all the single cells are pooled into a “library” and sequenced using conventional, short-read, Illumina sequencing technology. Computational analysis then sorts all those DNA reads that originate from each of the single cells encapsulated in the reaction vessel. This allows one to build a unique RNA expression pattern for each cell, and by using sophisticated biostatistical analysis, one can assign cells having similar patterns or “profiles” into common groups. Using this methodology, the various types of cells present in a solid tumor mass can be identified based purely on the RNA made in each of the cell types. This information can be used to understand tumor metastasis, develop predictive biomarkers of disease progression and aid in novel drug development.
To gain insight into how your colleagues are using single cell solutions to drive discovery with gene expression, immune, and copy number profiling you are invited to attend a Single Cell Analysis Mini Symposium on Wednesday November 7 from 2:00 t0 5:30 PM in the Cancer and Genetics Research Complex Auditorium. The meeting is free to attend, but registration is required (click here to register). The symposium is a joint event brought to you by the ICBR, 10x Genomics, Miltenyi Biotec, and Illumina. Come learn about 10x Genomics, Miltenyi, and Illumina solutions and methodologies for single cell studies, including cell viability, sequencing depth, and more.
If you are interested in utilizing the Chromium technology please contact Dr. Yanping Zhang, Scientific Director, ICBR Gene Expression & Genotyping Core or Dr. David Moraga (email/phone) Scientific Director, ICBR Next Generation Sequencing Core.