Bonnie L. Brown

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

The Cost of Nutrient Reduction: A Case Study of Chesapeake Bay

Over

Effects of age and composition of field-produced biofilms on oyster larval setting

3.5 billion were spent toward nutrient controls in the Chesapeake Bay watershed between 1985 and 1996. These expenditures were based on cost-shares between federal, state, local, and private sources and should be considered conservative. A comparison of point and nonpoint nutrient control expenditures in the Bay basin showed that about 50% of the funds were directed toward agricultural best management practices and 45% were allocated for point source nutrient reductions and combined sewer overflows (20% and 25%, respectively). The remainder was spent on various other estuarine-targeted programs. Maryland was responsible for 79% of the total expenditures, 16% was expended by Virginia, and the remainder was attributable to the District of Columbia and Pennsylvania (4% and 1%, respectively). The most costly basin was the Patuxent (over

MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry

125 per kilogram of total nitrogen removed) and the least costly was the Eastern Shore of Virginia, where the cost was just under

Characterization of human DNA in environmental samples

21 per kilogram of nitrogen removed. ...Over

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach

3.5 billion were spent toward nutrient controls in the Chesapeake Bay watershed between 1985 and 1996. These expenditures were based on cost-shares between federal, state, local, and private sources and should be considered conservative. A comparison of point and nonpoint nutrient control expenditures in the Bay basin showed that about 50% of the funds were directed toward agricultural best management practices and 45% were allocated for point source nutrient reductions and combined sewer overflows (20% and 25%, respectively). The remainder was spent on various other estuarine-targeted programs. Maryland was responsible for 79% of the total expenditures, 16% was expended by Virginia, and the remainder was attributable to the District of Columbia and Pennsylvania (4% and 1%, respectively). The most costly basin was the Patuxent (over

Mitochondrial DNA mixed-stock analysis of American shad: Coastal harvests are dynamic and variable

125 per kilogram of total nitrogen removed) and the least costly was the Eastern Shore of Virginia, where the cost was just under

Resource Availability Effects on Nitrate-Reducing Microbial Communities in a Freshwater Wetland

21 per kilogram of nitrogen removed. For the control practices examined, the analysis found that nutrient management was the least costly. Animal waste control and low tillage were the second and third most economical nutrient management strategies. Significant reductions were achieved from point sources for both total nitrogen (16%) and total phosphorus (53%) by 1996. Virtually all of the large reduction in point source phosphorus was a result of the phosphorus detergent ban implemented basinwide in the late 1980s. Nonpoint source nutrient reductions were more modest. An approximate 12% reduction in phosphorus was accompanied by an 8% reduction in total nitrogen loadings. Despite these nutrient reductions, no significant improvements in bottom-dissolved oxygen levels were detected along the Bay mainstem during the warmer months, and the mouth of Chesapeake Bay showed marginally significant degradation during the 11-year period. It was determined that dissolved oxygen conditions were influenced more by nitrogen than phosphorus reductions and that nutrient controls aimed at the mid-Bay region had the greatest potential for improving low dissolved oxygen conditions in the Bays bottom waters.

GENETIC STRUCTURING OF RELICT POPULATIONS OF GAPPER'S RED-BACKED VOLE (CLETHRIONOMYS GAPPERI)

Lack of success in restoring the native Eastern oyster, Crassostrea virginica, to Chesapeake Bay has been linked to the low occurrence of oyster larval setting in tributaries to the Bay. Among the many potential factors that could affect efforts to produce oysters through aquaculture or supplementation of shell beds is substratum condition. The present study examined larval setting on field-produced biofilms from Little Wicomico River (Virginia, USA) to assess whether bacterial community structure (examined by terminal restriction fragment length polymorphism, T-RFLP) or other characteristics of contemporary biofilms in this tributary (biofilm age and mass, algal abundance, and percentage organic matter) inhibited setting of larval oysters. The structure of the natural and heterogenous bacterial community in the biofilms and the success of oyster set were correlated, suggesting that specific microbial species may play a role in oyster setting. Larval set increased with biofilm age and mass, suggesting that established field-produced biofilms have no inhibitory effect. In contrast, the percentage of organic matter was negatively correlated with oyster set, whereas chlorophyll a concentration had no observed effect. The study expands prior knowledge by providing a more realistic timeframe for biofilm development (weeks as opposed to days), recounting effects of biofilms that are more representative of the natural dynamic and disturbance processes that would be expected to occur on submerged structures, and by incorporating seasonal and spatial variation. An important negative effect observed during the study period was heavy predation by Stylochus ellipticus on newly set oysters. Overall, the results of this study, which is the first assessment of the effects of biofilms produced naturally within a Chesapeake Bay tributary, suggest that the absence of large numbers of oysters in Little Wicomico River is not related to microbes or other specific characteristics of biofilms that develop on suitable setting substrata, but rather to heavy predation of newly set larvae.

Isolation and Characterization of Novel Microsatellite Markers for Yellow Perch (Perca flavescens)

Background: Long-read sequencing is rapidly evolving and reshaping the suite of opportunities for genomic analysis. For the MinION in particular, as both the platform and chemistry develop, the user community requires reference data to set performance expectations and maximally exploit third-generation sequencing. We performed an analysis of MinION data derived from whole genome sequencing of Escherichia coli K-12 using the R9.0 chemistry, comparing the results with the older R7.3 chemistry. Methods: We computed the error-rate estimates for insertions, deletions, and mismatches in MinION reads. Results: Run-time characteristics of the flow cell and run scripts for R9.0 were similar to those observed for R7.3 chemistry, but with an 8-fold increase in bases per second (from 30 bps in R7.3 and SQK-MAP005 library preparation, to 250 bps in R9.0) processed by individual nanopores, and less drop-off in yield over time. The 2-dimensional (“2D”) N50 read length was unchanged from the prior chemistry. Using the proportion of alignable reads as a measure of base-call accuracy, 99.9% of “pass” template reads from 1-dimensional (“1D”) experiments were mappable and ~97% from 2D experiments. The median identity of reads was ~89% for 1D and ~94% for 2D experiments. The total error rate (miscall + insertion + deletion ) decreased for 2D “pass” reads from 9.1% in R7.3 to 7.5% in R9.0 and for template “pass” reads from 26.7% in R7.3 to 14.5% in R9.0. Conclusions: These Phase 2 MinION experiments serve as a baseline by providing estimates for read quality, throughput, and mappability. The datasets further enable the development of bioinformatic tools tailored to the new R9.0 chemistry and the design of novel biological applications for this technology. Abbreviations: K: thousand, Kb: kilobase (one thousand base pairs), M: million, Mb: megabase (one million base pairs), Gb: gigabase (one billion base pairs).