Presented by Lionel Guy
Lionel Guy and Thijs Ettema
Department of Cell and Molecular Biology and SciLifeLab, Uppsala University, Uppsala, Sweden
Only a few percent of prokaryotes are cultivable: the vast majority of them remains to be discovered, but recent technological developments in single-cell genomics now grant us access to this unknown diversity. To answer questions about microbial diversity and even how complex life forms emerged, we sample novel, deep-rooting taxa from a wide variety of environments.
Cells extracted from samples are sorted in 384-well plates using fluorescence-activated cell sorting (FACS). They are lysed, and their genome is amplified with multiple displacement amplification (MDA), yielding a single-cell amplified genome (SAG). SAGs representing cells of interest are multiplexed and sequenced with next-generation sequencing. Up to 100 SAGs can be sequenced on one lane of a Illumina HiSeq2500, and we plan to sequence several thousand SAGs every year.
The Galaxy platform was chosen to handle the vast amount of data generated in the single-cell genomics pipeline. The platform will be integrated in our LIMS system, keeping track of all samples and SAGs present in our lab. Three pipelines are being designed: the first takes raw reads from the sequencing runs as input, performs quality and contamination checks, assembles SAGs, and outputs scaffolds. The second is dedicated to physical and functional annotation of genomes. The third will perform comparative genomics, assemble sets of orthologous genes from our SAGs and published genomes, and perform phylogenomics on the aligned sequences, yielding high-quality phylogenetic trees.
By pairing single-cell genomics with advanced bioinformatics, we aim to shed some light on the deep roots of the Tree of Life.