Challenges of metagenomics and single-cell genomics approaches for exploring cyanobacterial diversity

Abstract

Cyanobacteria have played a crucial role in the history of early earth and continue to be instrumental in shaping our planet, yet applications of cutting edge technology have not yet been widely used to explore cyanobacterial diversity. To provide adequate background, we briefly review current sequencing technologies and their innovative uses in genomics and metagenomics. Next, we focus on current cell capture technologies and the challenges of using them with cyanobacteria. We illustrate the utility in coupling breakthroughs in DNA amplification with cell capture platforms, with an example of microfluidic isolation and subsequent targeted amplicon sequencing from individual terrestrial thermophilic cyanobacteria. Single cells of thermophilic, unicellular Synechococcus sp. JA-2-3-B'a(2-13) (Syn OS-B') were sorted in a microfluidic device, lysed, and subjected to whole genome amplification by multiple displacement amplification. We amplified regions from specific CRISPR spacer arrays, which are known to be highly diverse, contain semi-palindromic repeats which form secondary structure, and can be difficult to amplify. Cell capture, lysis, and genome amplification on a microfluidic device have been optimized, setting a stage for further investigations of individual cyanobacterial cells isolated directly from natural populations.

Recent advances and application in whole‐genome multiple displacement amplification

“…Davison et al . [86] obtained data from CRISPR spacers after amplification of single‐cell genomes of cyanobacteria with different growth temperatures and combined the reference sequences of the host and viruses to study the mechanisms of host/virus co‐evolution. Similarly, in 2019, Tu et al .…”

“…One example is the frequent application of MDA in the study of cyanobacteria diversity. Davison et al [86] obtained data from CRISPR spacers after amplification of single-cell genomes of cyanobacteria with different growth temperatures and combined the reference sequences of the host and viruses to study the mechanisms of host/virus co-evolution. Similarly, in 2019, Tu et al proposed a pervasive metagenome construction and analysis pipeline to provide insights into the reciprocal relationships within Microcystis colonies and cyanobacterial bloom mechanisms [87].…”

A Metagenomic Approach to Cyanobacterial Genomics

“…Single-cell genomics has been recently reviewed as an alternative approach for sequencing cyanobacteria in natural environments (Davison et al, 2015). This strategy consists basically of the capture of a single-cell followed by routines that are similar to the ones employed for axenic strains.…”

DRAGoM: Classification and Quantification of Noncoding RNA in Metagenomic Data

“…ncRNAs can also catalyze biochemical reactions (ribozymes) ( Doherty and Doudna, 2001 ), as exemplified by the well-known ribosomal RNAs (which catalyze protein synthesis) and group I and II introns (which catalyze the excision of themselves from the transcript) ( Adams et al, 2004a , b ). With the prevalence of metagenomics ( Virgin and Todd, 2011 ; Huttenhower et al, 2012 ; Shokralla et al, 2012 ; Williamson and Yooseph, 2012 ; Davison et al, 2015 ; Quince et al, 2017 ), more microbial genomic sequences, including the previously uncharacterized ones, have been accumulated into public databases. The amazing richness of microbial genomic data renders a great opportunity to study ncRNA.…”