In 1670, Antony van Leewenhoek made it possible to visualise a single cell under a microscope, now we have the potential to examine the entire genetic make-up of hundreds of single cells simultaneously.
Advances in next-generation sequencing (NGS) methods have enabled whole transcriptome sequencing of single cells in high-throughput. The main limitation of sequencing is cost. It is true that the cost of sequencing has now decreased, but this has not been the case for the library preparation step. If this part of the process could be miniaturised then the cost of library preparation could also be reduced. This sounds simple until you have tried to pipette very small volumes of reagents and samples using manual or large volume automated systems.
We have found the answer – reproducible, low-cost, low-volume sequencing on the benchtop!
At SLAS2016 international conference, San Diego (January 23-27th 2016) Dr. Louise Laurent (University of California, San Diego) will discuss how TTP Labtech’s mosquito® nanolitre liquid handler has enabled new strategies to be developed for low-cost single-cell transcriptome sequencing.
In this talk, Louise will present results indicating that combining the C1 Single-Cell RNA Seq Auto Prep System (Fluidigm Corp., US) for cDNA production, along with mosquito HTS (TTP Labtech) reproducibly yields high-quality single-cell RNAseq libraries at reaction volumes down to 2 ul, which can be used to identify biological differences between single cells.
The C1 Single-Cell RNA Seq Auto Prep System provides automated separation of single cells into individual microchambers where the reverse transcription reaction using SMARTer template-switching technology (Clontech Laboratories, Inc., US) generates high-quality cDNA. Using mosquito HTS, as little as 20 pg of cDNA was used as input into the Nextera XT library preparation kit (Illumina).
mosquito HTS employs true positive-displacement liquid transfer to handle nanolitre to microlitre (25 nL – 1.2 µL) volumes quickly and accurately in a cross-contamination free manner. The results demonstrate that this approach can be used for automated high-throughput generation of high quality RNA sequencing libraries from extremely low sample inputs.
Other examples of using mosquito liquid handlers in single-cell genomic applications (both RNA and gDNA) are detailed in our recent application note from Stephen Quake and Irving Weissman’s labs. One study used this miniaturisation method to identify novel bacterial species from environmental metagenomic samples. TTP Labtech’s mosquito X1 and HTS were used for normalisation and library prep at low volumes. In the second study, RNA expression was examined in single-cell analysis of macrophages from healthy and abnormal mouse tissues. The total DNA (gDNA or cDNA) required for these studies was reduced down 60 – 80pg.
Source – TTP Labtech