eDNAFlow, an automated, reproducible and scalable workflow for analysis of environmental DNA sequences exploiting Nextflow and Singularity.

Metabarcoding of environmental DNA (eDNA) when coupled with high throughput sequencing is revolutionising the way biodiversity can be monitored across a wide range of applications. However, the large number of tools deployed in downstream bioinformatic analyses often places a challenge in configuration and maintenance of a workflow, and consequently limits the research reproducibility. Furthermore, scalability needs to be considered to handle the growing amount of data due to increase in sequence output and the scale of project. Here, we describe eDNAFlow, a fully automated workflow that employs a number of state-of-the-art applications to process eDNA data from raw sequences (single-end or paired-end) to generation of curated and noncurated zero-radius operational taxonomic units (ZOTUs) and their abundance tables. This pipeline is based on Nextflow and Singularity which enable a scalable, portable and reproducible workflow using software containers on a local computer, clouds and high-performance computing (HPC) clusters. Finally, we present an in-house Python script to assign taxonomy to ZOTUs based on user specified thresholds for assigning lowest common ancestor (LCA). We demonstrate the utility and efficiency of the pipeline using an example of a published coral diversity biomonitoring study. Our results were congruent with the aforementioned study. The scalability of the pipeline is also demonstrated through analysis of a large data set containing 154 samples. To our knowledge, this is the first automated bioinformatic pipeline for eDNA analysis using two powerful tools: Nextflow and Singularity. This pipeline addresses two major challenges in the analysis of eDNA data; scalability and reproducibility.

Atomic force microscopy with integrated on-chip interferometric readout.

The most common readout technique used in atomic force microscopy (AFM) is based on optical beam deflection (OBD), which relies on monitoring deflection of the cantilever probe by measuring the position of the laser beam reflected from the free end of the AFM cantilever. Although systems using the OBD readout can achieve subnanometre displacement resolution and video rate imaging speeds, its main limitation is size, which is difficult to minimise, thus limiting multiprobe imaging capability. Currently, system miniaturisation has been accommodated by adopting on-chip electrical readout solutions, often at the expense of measurement sensitivity. To date, no cost-effective AFM readout solution exists without sacrificing either measurement sensitivity, system miniaturisation, or multiprobe array scalability. In this paper we present an AFM probe with integrated on-chip optical interferometric readout based on silicon photonics. Our AFM probe combines the advantages of subnanometre resolution of optical readouts with on-chip miniaturisation. The adopted approach determines deflection of the cantilever using an integrated on-chip photonics waveguide by monitoring the separation between the sensing cantilever and an interrogating grating. The implemented methodology provides ultimate interferometric resolution and sensitivity, on-chip miniaturisation, and array scalability, which makes possible ultrafast multiprobe-array AFM imaging. Using a Digital Instruments D3000 AFM retrofitted with our cantilever probe and integrated readout, we report sub-nanometre AFM topography images obtained on reference samples. We demonstrate RMS static AFM noise level of 19 pm, outperforming the operation of this system in its standard, optical beam deflection configuration (51 pm). The noise spectrum measurements of our probe indicate that the integrated readout is shot noise limited, achieving a deflection noise density (DND) of 36fm/Hz.

Multimodal atomic force microscopy with optimized higher eigenmode sensitivity using on-chip piezoelectric actuation and sensing.

Atomic force microscope (AFM) cantilevers with integrated actuation and sensing provide several distinct advantages over conventional cantilever instrumentation. These include clean frequency responses, the possibility of down-scaling and parallelization to cantilever arrays as well as the absence of optical interference. While cantilever microfabrication technology has continuously advanced over the years, the overall design has remained largely unchanged; a passive rectangular shaped cantilever design has been adopted as the industry wide standard. In this article, we demonstrate multimode AFM imaging on higher eigenmodes as well as bimodal AFM imaging with cantilevers using fully integrated piezoelectric actuation and sensing. The cantilever design maximizes the higher eigenmode deflection sensitivity by optimizing the transducer layout according to the strain mode shape. Without the need for feedthrough cancellation, the read-out method achieves close to zero actuator/sensor feedthrough and the sensitivity is sufficient to resolve the cantilever Brownian motion.