Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples

Background: Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms recently implemented in Oxford Nanopore's own control software. By examining the first few hundred bases of a DNA molecule as it passes through a pore, software can determine if the molecule is sufficiently interesting to sequence in its entirety. If not, the molecule is ejected from the pore by reversing the voltage across it, freeing the pore for a new molecule. User supplied sequences define the targets to be sequenced or ejected. Here we explore the potential of using adaptive sampling for enrichment of rarer species within metagenomic samples. Results: We created a synthetic mock community consisting of seven bacterial species at different proportions ranging from 1.2% to 47% and used this as the basis for a series of enrichment and depletion experiments. To investigate the effect of DNA length on adaptive sampling efficiency, we created sequencing libraries with mean read lengths of 1.7 kbp, 4.7 kbp, 10.6 kbp, and 12.8 kbp and enriched or depleted for individual and multiple species over a series of sequencing runs. Across all experiments enrichment ranged from 1.67-fold for the most abundant species with the shortest read length to 13.87-fold for the least abundant species with the longest read length. Factoring in the reduction to sequence output associated with repeatedly rejecting molecules reduces the calculated efficiency of this enrichment to between 0.96-fold and 4.93-fold. We note that reducing ejections due to false negatives (approximately 36%) would significantly increase efficiency. We used the relationship between abundance, molecule length and enrichment factor to produce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlated strongly with experimental data. A web application is provided to allow researchers to explore model predictions in advance of performing their own experiments. Conclusions: Our data clearly demonstrates the benefit for enriching low abundant species in adaptive sampling metagenomic experiments, especially with longer molecules, and our mathematical model can be used to determine whether a given experimental DNA sample is suitable for adaptive sampling. Notably, repeated voltage reversals have no effect on pore stability.

Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates

Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune response, insulin secretion, and body temperature regulation. It is activated by cytosolic ADP ribose (ADPR) and contains a nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain homologous to ADPR phosphohydrolases (ADPRases). Human TRPM2 (hsTRPM2) is catalytically inactive due to mutations in the conserved Nudix box sequence. Here, we show that TRPM2 Nudix motifs are canonical in all invertebrates but vestigial in vertebrates. Correspondingly, TRPM2 of the cnidarian Nematostella vectensis (nvTRPM2) and the choanoflagellate Salpingoeca rosetta (srTRPM2) are active ADPRases. Disruption of ADPRase activity fails to affect nvTRPM2 channel currents, reporting a catalytic cycle uncoupled from gating. Furthermore, pore sequence substitutions responsible for inactivation of hsTRPM2 also appeared in vertebrates. Correspondingly, zebrafish (Danio rerio) TRPM2 (drTRPM2) and hsTRPM2 channels inactivate, but srTRPM2 and nvTRPM2 currents are stable. Thus, catalysis and pore stability were lost simultaneously in vertebrate TRPM2 channels.

Karakteristik Pori dan Hubungannya dengan Permeabilitas pada Tanah Vertisol Asal Jeneponto Sulawesi Selatan

This study aims to determine the characteristics of soil pores and therelationship of pore characteristics with soil permeability. Soil samples were collected from Punagaya Village, Bangkala District, Jeneponto Regency, South Sulawesi. Soil samples were taken from 20 observation points representing two types of land use ie cultivated land and fallow field at a depth of 0-60 cm. Soil analysis (pH, organic material, C-organic, cation exchange capacity, bulk density and soil texture), Pore characteristics(porosity, pore distribution and pore stability), and permeability. Analysis of soil properties and pore characteristics was descriptive, while the relationship between pore characteristics and permeability was analyzed by single or multiple regression and correlation analysis. The results showed that the permeability of soil vertisol 0.29 cm/h (slow) and the average value of soil pore characteristics of vertisol as follows: porosity ranged 56.49%, fast drainage pore 4.35%, water pore available 17.8% And 32.5% micro pores, and 36.46% pore stability. Based on the regression and correlation test, it is known that Porosity and porosity and pore stability index have the greatest influence on permeability.