Metagenomic analysis of a mega-city river network reveals microbial compositional heterogeneity among urban and peri-urban river stretch

MinION Nanopore Sequencing Accelerates Progress towards Ubiquitous Genetics in Water Research

“…For the tissue of aquatic organisms, biomass concentration will be unnecessary [32,34,59]. Genetic material from biomass-rich sediment samples (0.2 to 1 g) and sludge samples (0.5 mL) can also be extracted directly [45,51,60,61,75], or after centrifugation of small to medium slurry volumes (2 to 50 mL) with collection of the pellets [52,62,69]. Zooplanktonic biomass can be harvested with meshes having 200 µm openings [36], while the biomass of algae [74] and cyanobacteria [48] can be isolated by medium to large volume centrifugation (0.05 to 1 L).…”

“…The related procedures involve sample homogenization and agitation in bead tubes with the addition of a cell lysis solution, phase separation, DNA or RNA binding, and washing steps before the purified genetic material can be obtained. Most studies working with water samples used the respective kits, while kits optimized for soil were used to extract sediment [46,49,53,60,61,67], wastewater [62,69,70,72], and algal biomass samples [59]. Enzymes like lysozymes sometimes assist with cell lysis, for example in a study of planktonic riverine microbial consortia [37] and plastic-associated species in the Mediterranean Sea [44].…”

“…As a minimum, the purified genetic material needs repair and preparation of the fragmented DNA/RNA strand ends followed by ligation (i.e., attachment) of the sequencing adaptors which interact with the flow cell nanopores. This can be done with ligation sequencing kits that were popular choices to sequence DNA [43,45,46,49,52,53,55,60,61,67,70,72,76] and derived amplicons [31,32,34,35,44,58,59,73,74], or amplicons of complementary DNA (cDNA, see below) [28]. Semmouri et al [33] evaluated the MinION potential for direct RNA nanopore sequencing using the corresponding kit.…”

“…ONT's 16S barcoding kit enables multiplexing of up to 24 samples. Native barcoding kits enable multiplexing of up to 96 samples, either without PCR [45,49,55,60,61,67,75], or following a PCR step [42,69]. Specialist kits for PCR-based barcoding expansion were used in some of the reviewed applications [34,59].…”

“…Guppy includes such post-processing features, i.e., barcoding/demultiplexing, adapter trimming, and alignment. Other popular application choices in the reviewed studies include qcat [83] for demultiplexing (i.e., to assign reads to their barcodes when several barcoded samples are run on a single flow cell) [44,45,[59][60][61]67,76] and Porechop [84] for adaptor trimming [45,46,48,52,55,58,[60][61][62]64,67,69,72,74,76]. However, qcat and Porechop are no longer supported, and the use of Guppy is recommended.…”