Replicate DNA metabarcoding can discriminate seasonal and spatial abundance shifts in river macroinvertebrate assemblages.

The delivery of consistent and accurate fine-resolution data on biodiversity using metabarcoding promises to improve environmental assessment and research. Whilst this approach is a substantial improvement upon traditional techniques, critics note that metabarcoding data are suitable for establishing taxon occurrence, but not abundance. We propose a novel hierarchical approach to recovering abundance information from metabarcoding, and demonstrate this technique using benthic macroinvertebrates. To sample a range of abundance structures without introducing additional changes in composition, we combined seasonal surveys with fish-exclusion experiments at Catamaran Brook in northern New Brunswick, Canada. Five monthly surveys collected 31 benthic samples for DNA metabarcoding divided between caged and control treatments. A further six samples per survey were processed using traditional morphological identification for comparison. By estimating the probability of detecting a single individual, multispecies abundance models infer changes in abundance based on changes in detection frequency. Using replicate detections of 184 genera (and 318 species) from metabarcoding samples, our analysis identified changes in abundance arising from both seasonal dynamics and the exclusion of fish predators. Counts obtained from morphological samples were highly variable, a feature that limited the opportunity for more robust comparison, and emphasizing the difficulty standard methods also face to detect changes in abundance. Our approach is the first to demonstrate how quantitative estimates of abundance can be made using metabarcoding, both among species within sites as well as within species among sites. Many samples are required to capture true abundance patterns, particularly in streams where counts are highly variable, but few studies can afford to process entire samples. Our approach allows study of responses across whole communities, and at fine taxonomic resolution. We discuss how ecological studies can use additional sampling to capture changes in abundance at fine resolution, and how this can complement broad-scale biomonitoring using DNA metabarcoding.

Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets.

BACKGROUND: Pseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for nuclear mitochondrial DNA segments (nuMTs) in large COI datasets. We do this by: (1) describing gene and nuMT characteristics from an artificial COI barcode dataset, (2) show the impact of two different pseudogene removal methods on perturbed community datasets with simulated nuMTs, and (3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile analysis were used to detect pseudogenes. RESULTS: Our simulations showed that it was more difficult to identify nuMTs from shorter amplicon sequences such as those typically used in metabarcoding compared with full length DNA barcodes that are used in the construction of barcode libraries. It was also more difficult to identify nuMTs in datasets where there is a high percentage of nuMTs. Existing bioinformatic pipelines used to process metabarcode sequences already remove some nuMTs, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove up to 5% of sequences even when other filtering steps are in place. CONCLUSIONS: Open reading frame length filtering alone or combined with hidden Markov model profile analysis can be used to effectively screen out apparent pseudogenes from large datasets. There is more to learn from COI nuMTs such as their frequency in DNA barcoding and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI nuMTs to public databases to facilitate future studies.

Comparing the microbial inhibition of nanofibres with multi-metal ion exchanged nano-zeolite Y in air sampling.

AIMS: Fibre membranes containing metals have attracted great attention because of their high antibacterial efficiency. However, comparison of antibacterial activity of fibres with multi-metals in air samples has remained understudied. METHODS AND RESULTS: Different ion exchanged nano-zeolite Y (IE-NZY) of Ag, Zn and Cu was studied. Polyvinylpyrrolidine/polyvinylidene fluoride nanofibres containing various IE-NZY were synthesized according to electrospinning technique. The presence of metal ions was confirmed using XRF. The morphological properties of nanofibres were characterized by SEM. Zone of inhibition was seen between 10·1 and 12 mm against Staphylococcus aureus and 11·5-14·57 for Escherichia coli. IE-NZY containing Ag, Zn and Cu had the highest antibacterial efficiency. In the air samples, there were any colonies on the media under the Ag/Cu-NZY and Zn/Cu/Ag-NZY nanofibres. CONCLUSIONS: The bacterial inhibition for nanofibres containing a three metal nano-zeolite Y (TM-NZY) is higher than bimetals (BM-NZY) types while for monometals nano-zeolite Y (MM-NZY), it was lower compared to the others. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate significant antibacterial activity of ion-exchanged NZY in air sampling.

Comprehensive biodiversity analysis via ultra-deep patterned flow cell technology: a case study of eDNA metabarcoding seawater.

The characterization of biodiversity is a crucial element of ecological investigations as well as environmental assessment and monitoring activities. Increasingly, amplicon-based environmental DNA metabarcoding (alternatively, marker gene metagenomics) is used for such studies given its ability to provide biodiversity data from various groups of organisms simply from analysis of bulk environmental samples such as water, soil or sediments. The Illumina MiSeq is currently the most popular tool for carrying out this work, but we set out to determine whether typical studies were reading enough DNA to detect rare organisms (i.e., those that may be of greatest interest such as endangered or invasive species) present in the environment. We collected sea water samples along two transects in Conception Bay, Newfoundland and analyzed them on the MiSeq with a sequencing depth of 100,000 reads per sample (exceeding the 60,000 per sample that is typical of similar studies). We then analyzed these same samples on Illumina's newest high-capacity platform, the NovaSeq, at a depth of 7 million reads per sample. Not surprisingly, the NovaSeq detected many more taxa than the MiSeq thanks to its much greater sequencing depth. However, contrary to our expectations this pattern was true even in depth-for-depth comparisons. In other words, the NovaSeq can detect more DNA sequence diversity within samples than the MiSeq, even at the exact same sequencing depth. Even when samples were reanalyzed on the MiSeq with a sequencing depth of 1 million reads each, the MiSeq's ability to detect new sequences plateaued while the NovaSeq continued to detect new sequence variants. These results have important biological implications. The NovaSeq found 40% more metazoan families in this environment than the MiSeq, including some of interest such as marine mammals and bony fish so the real-world implications of these findings are significant. These results are most likely associated to the advances incorporated in the NovaSeq, especially a patterned flow cell, which prevents similar sequences that are neighbours on the flow cell (common in metabarcoding studies) from being erroneously merged into single spots by the sequencing instrument. This study sets the stage for incorporating eDNA metabarcoding in comprehensive analysis of oceanic samples in a wide range of ecological and environmental investigations.

Inpatient paediatric use of intravenous immunoglobulin at an academic medical centre.

INTRODUCTION: Intravenous immunoglobulin (IVIG) is an important research topic because of its efficacy in the management of an increasing number of diseases, its high cost and limited availability. This study was designed to evaluate the paediatric inpatient use of IVIG and identify strategies to reduce the drug expenditures. METHODS: Over a six-month period, physician and nursing charts, and notes for subjects who were treated with IVIG, were reviewed to gather the required data. This included patient demographics, IVIG, indications, dosage regimen, adverse drug reactions (ADRs) and their management. RESULTS: 58.3 percent of IVIG infusions were ordered for labelled indications. Patients in the labelled group experienced more clinical improvement than subjects in the off-label group. Haematologists and neurologists were the most prevalent prescribers. ADRs were more prevalent in the off-label group. Hypotension, fever, headache and chills were the most common adverse effects. ADRs were managed with drugs in 22.9 percent of IVIG administrations and IVIG infusions were modified in 12.5 percent of infusions. CONCLUSION: ADRs were more prevalent in this hospital than those reported by other authors. This may be due to nursing negligence of the recommended infusion rate, higher sensitivity of our population or to the brands of IVIG which are used in the hospital. This shows the need for further evaluation of IVIG prescription and administration.