Single‐cell genomics based on Raman sorting reveals novel carotenoid‐containing bacteria in the Red Sea

Abstract

SummaryCell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.

Raman‐activated cell sorting and metagenomic sequencing revealing carbon‐fixing bacteria in the ocean

“…S1). In our previous design, the slide with cell sample had to be manually flipped over for cell ejection (Song et al, 2017). This procedure increases the risk of contamination for single-cell DNA amplification and slows down the sorting process and represents a hurdle for automation.…”

“…Raman spectroscopy has been proved to be a useful approach which could provide comprehensive phenotype information of single cells in a non‐destructive manner. More importantly, by combining with the stable isotope probing method which is using stable isotope substituted substrates for keeping normal metabolism of cells, Raman‐SIP provides a promising tool for pinpointing specific metabolic functions of certain species within a complex community (Huang et al ., ; Li et al ., b; Wang et al ., ; Zhang et al ., a; Song et al ., ) or general metabolic activity (Berry et al ., ; Wang et al ., b; Tao et al ., ) at single‐cell level. Given above merits, Raman activated cell sorting and sequencing could undisputedly offer a direct link between phenotype and genotype of cells, which is especially significant in microbiology.…”

“…Several Raman activated cell sorting methods have been established, including Raman tweezers, RACE (Song et al, 2017), microfluidic flow sorting (RACS, RADS) (Zhang et al, 2015b;McIlvenna et al, 2016;Wang et al, 2017). RACE with single cell genomics can provide direct link between phenotype and genotype of the cell.…”

“…Raman-stable isotope probing (Raman-SIP) has been employed to probe bacterial function at the single cell level (Huang et al, 2009;Bankevich et al, 2012;Li et al, 2012b;Wang et al, 2016a). To link the function to its underlying genotypes, we have developed a series of Raman-Activated Cell Sorting (RACS) techniques, such as Raman-activated cell ejection (RACE) and Raman-activated microfluidic sorting (RAMS) (Zhang et al, 2015b;McIlvenna et al, 2016;Song et al, 2017), to isolate individual cells based on their SCRS characteristics. After RACS, genomic DNA from single cells was amplified by multiple displacement amplification (MDA) and subsequently processed for sequencing (Song et al, 2017).…”

Fingerprinting microbiomes towards screening for microbial antibiotic resistance

“…Also, with the non-destructive character of bio-spectroscopic fast screening reducing the amount of interrogating targets, many other techniques can be attached for further investigation, e.g., single cell sorting 54,57 , -omics 40 , etc. One good example can be found in Huang's work describing novel marine strain identification via Raman spectroscopy coupled with single-cell genomics 123 . Raman spectra are also reported in morphology-specific genomic analysis of human tissues combined with microdissection sequencing 124 or characterizing metabolic alterations in mouse liver coupled with metabolomics and transcriptomics 125 .…”