Category: Micro- and Nanotechnologies
There has been considerable recent interest in sequencing single cells, and several lab-made and commercial solutions have emerged, particularly for 3’ end sequencing of mRNA. Microfluidic reactors, nanoliter volume open wells, and emulsion droplet-based methods are available. Individual commercial offerings however, typically combine hardware and reagents to run a specific protocol. In pursuit of greater flexibility, we have developed methods based on nanoliter volume plates, picoliter spotters, microscopes, and reagents, all from different vendors. Open nano-wells allow multiple sequential reagent additions and also allow microscopic inspection of the cells for surface markers or live-dead status. Relative concentrations of nucleic acids and reagents are preserved with reaction volumes 1000X smaller than conventional bulk reactions, and this appears to preserve reaction efficiency.
With this system we have developed a tagmentation-based whole genome sequencing protocol that does not require pre-amplification. Physical coverage has been observed up to 0.1x per cell and as a ensemble, the data reproduces the evenness of coverage and GC bias typical of bulk genome libraries that use a PCR step. This enables grouping of subclones with in a mixed cellular population such as a heterogeneous tumour. Indexes are applied in row-column format so 72+72 indices can uniquely identify up to 5184 cells. The current practical limit is about 3000 cells per run though this may readily rise with faster spotting. The authors, working in parallel in two laboratories have built and sequenced nearly 40,000 single cell whole genome libraries in the last 18 months.
A whole transcript single cell sequencing protocol has also been developed. Initial results show >3000 genes per cell with at greater than ten reads and ensembles of cells show similar coverage to bulk sequence. The flexibility of this open platform allows us to consider other sample preparation methods for epigenomics, and small RNAs as well as combinatorial methods which could interrogate hundreds of thousands of cells.Results from the whole genome and transciptome sequencing methods will be presented, showing the degree to which this flexible and highly robust platform represents a compelling way forward in single cell genomics analysis.
Robin Coope– Group Leader, Instrumentation, BC Cancer Genome Sciences Centre, Vancouver, BC, Canada