Webinar on demand – High coverage single-cell metagenomics using PTA

High coverage single-cell metagenomics using PTA

Single-cell metagenomic sequencing (scMetaG) can provide maximum-resolution insights into complex microbial communities. However, library preparation in multiwell plates is prohibitively expensive and does not reach the cell counts that are typical for modern droplet based approaches. While microfluidic droplet approaches have been very successful for protocols where enzymes and lysis buffers can be delivered as a one-pot solution, this method is not applicable to multiple-step WGS library preparation.

We overcome this by using semi-permeable capsules – two-phase polymer mixes – which contain the cells and enable exchange of buffers, enzymes and oligos, while trapping RNA and genomic DNA inside. We developed an optimized harsh lysis protocol for bacteria and adapted Multiple Displacement Amplification (MDA) as well as Primary Template-directed Amplification (PTA) for whole-genome amplification. The data of over 50k bacterial cells (mock community as well as human saliva) were analyzed using our Bascet/Zorn single-cell WGA pipeline (http://zorn.henlab.org). We show that PTA yields superior performance over MDA. We also characterized lysis biases and find that they are not simply driven by a gram-positive/negative dichotomy. Overall, Bascet and Zorn enable reproducible scMetaG analysis, allowing users to query microbiomes at unprecedented resolution and scale.

About the speaker
Johan Henriksson is a researcher and group leader at the Department of Molecular Biology at Umeå University, where his work combines single-cell technologies, multi-omics, bioinformatics and wet-lab method development. His research spans microbial single-cell analysis as well as T-cell biology and immunotherapy, with a strong focus on developing new experimental and computational approaches.