Microbial single cell genomics analyses

The Singe Cell Genomics Center (SCGC; https://scgc.bigelow.org) at the Bigelow Laboratory for Ocean Sciences is the only facility in the world capable of performing large-scale, integrated single-cell and single-viral-particle genome and phenome analyses on diverse environmental microbiomes. The processing of exceedingly small DNA quantities makes single cell genomics highly susceptible to DNA contamination and amplification biases. SCGC has developed techniques to monitor and minimize methodological artifacts at every step of its workflow. Cell sorting and DNA amplification are performed in a cleanroom, and all consumables are decontaminated using in-house methods. Control wells on each microplate are used to detect potential DNA contamination. To prevent index switching during DNA sequencing, multiplexed libraries contain dual 10 bp barcodes. Genome de novo assemblies are evaluated by diverse, in-house and external QC tools. The entire workflow is assessed for contamination and assembly errors using microbial benchmark cultures with diverse genome complexity and G+C content, indicating no non-target and undefined bases and average frequencies of mis-assemblies, indels, and mismatches at <5 per 100 kbp. The following SCGC services will be performed: 1. Generation of fluorescence-activated cell sorting (FACS) - based single amplified genomes (SAGs) (SCGC services S-201, S-202, and S-203). As part of these services, cells or other particles are separated by FACS, lysed, and their DNA is amplified in 384-well microplates. Cells/particles may be selected based on particle autofluorescence or fluorescent probes. Cells/particles are lysed and their DNA is denatured. Subsequently, the S-201 service uses WGA-X for genomic DNA amplification, a method developed by SCGC. Compared to the earlier versions of multiple displacement amplification technique (MDA), WGA-X improves average genome recovery from individual cells and viral particles, with most notable enhancements observed in SAGs with high G+C content. Service S-202 uses a genomic DNA amplification technique WGA-Y, which offers a further improvement in the average genome recovery from single cells. Service S-203 complements S-201 and S-202 by measuring oxygen respiration rates of individual microbial cells. Deliverables of FACS-based SAG generation services include: a. 384-well microplates containing WGA products of individual particles b. Phenotype measurements of sorted particles c. WGA kinetics in each well 2. Sequencing of FACS - based SAGs (services S-211, S-212, S-213, S-221, S-222, and S-223). These services include Illumina library preparation from SAG gDNA, shotgun sequencing, read demultiplexing, de novo assembly, functional and taxonomic annotation, and genome quality control. Deliverables include: a. raw sequence reads b. de novo SAG assemblies c. SAG functional annotation d. SAG taxonomic assignments e. general genome properties, such as GC content and coding density f. files to assist manual QC: outputs of tetramer PCA, BLASTn and checkM g. single particle reports. 3. Semi-permeable capsule (SPC) SAG generation and sequencing (services S-311, S-312, S-321, S-322, and S-323). Cells and other particles are compartmentalized into into SPCs using microfluidic platforms, then lysed, and their genomic DNA is amplified by WGA-Y to produce SPC SAGs. Environmental SPC SAGs are mixed with E. coli SPC SAGs, serving as internal standards. The SPC SAG DNA is combinatorially barcoded and then used in Illumina library preparation. After shotgun sequencing, reads are demultiplexed, de novo assembled, and the obtained assemblies are annotated and quality controlled. Deliverables include: h. barcoded SPC SAGs i. multiplexed Illumina libraries j. raw sequence reads k. de novo SAG assemblies l. SAG functional annotation m. SAG taxonomic assignments n. general genome properties, such as GC content and coding density o. files to assist manual QC 4. Complementary services: a. Handling of biohazard level 2 samples (S-004) b. FACS SAG re-arraying (S-005) c. Provision of cell cryoprotectant glyTE (S-009) d. Consultation (S-011) e. Customized services (S-020)