An updated genetic marker for detection of Lake Sinai Virus and metagenetic applications

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Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials & Methods

Sampling

Assay amplification conditions

Illumina amplicon sequencing

Clustering and quantification of variants

Compositional analysis

Results

Primer development and application to surveys

Metagenetic characterization of LSV diversity

Discussion

Conclusions

Supplemental Information

Visualization of the Lake Sinai Virus assay product by electrophoresis

Amplicons were migrated in 1.2% agarose. Positive samples are individual bee specimens collected from Lincoln, Nebraska. Lane 1: 100 bp deoxyribonucleic acid ladder/marker, Lane 2–5: samples scored negative, Lane 6: tick markers, Lane 7–10: samples scored positive, Lane 11: Negative control.

DOI: 10.7717/peerj.9424/supp-1

Pairwise correlations between technical replicates of sample UNL.VT.250

Scatterplot points represent the frequency of individual OTUs in each pair. Technical replicates are based on independent amplifications and library preparations from the same source sample.

DOI: 10.7717/peerj.9424/supp-2

Lake Sinai Virus nucleotide alignment used for primer selection

Bases are colorized to indicate overall conservation within the alignment. Location and orientation of each primer are indicated by arrows. Sequences were obtained from Cornman (2018).

DOI: 10.7717/peerj.9424/supp-3

Script used to tabulate LSV diversity within samples

The distribution of protein differences (unit increments) weighted by OTU frequency and relative to the most abundant OTU in each sample. The data files used as input are appended and commented out. The script is specific to the analysis described in the text and is not intended as a general tool.

DOI: 10.7717/peerj.9424/supp-4

Example sequences obtained from positive LSV assay amplicons

Sanger sequences from the USDA set. Maryland and California samples begin with MD and CA, respectively. F and R designate sequences obtained with forward and reverse primers, respectively. Sequence names indicate whether the sample was positive using LSV2-specific primers using the assay described in Traynor et al. (2016).

DOI: 10.7717/peerj.9424/supp-5

Representative sequences of Lake Sinai Virus operational taxonomic units (OTUs)

OTU clustering was performed at 98% sequence identity and denoised as described in the text. Sequence IDs are alphanumeric identifiers based on the underlying read from which the cluster representative derives.

DOI: 10.7717/peerj.9424/supp-6

Raw occurrence counts of each Lake Sinai Virus OTU in each sample

DOI: 10.7717/peerj.9424/supp-7

Additional Information and Declarations

Competing Interests

Jay D. Evans is an Academic Editor for PeerJ.

Author Contributions

Deborah D. Iwanowicz, Judy Y. Wu-Smart, Tugce Olgun, Autumn H. Smart, Clint R.V. Otto, Dawn Lopez conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Jay D. Evans conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Robert Cornman conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Field Study Permissions

The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):

No new sampling occurred for this study. Sites used for sampling are listed in the cited previous work. These consisted of both our own institutional sites as well as private and public lands. Private property was accessed with the expressed verbal permission of landowners and beekeepers. As the location and health status of colonies is protected trade information, access is typically given on condition of anonymity and cannot be disclosed to other parties. However, we are authorized to state that access to apiaries on private lands in North Dakota were given by Zac Browning, Browning Honey Company. Access to apiaries on private lands in South Dakota were given by Brett Adee, Adee Honey Farms.

Data Availability

The following information was supplied regarding data availability:

OTU clusters and abundances, Sanger-sequenced amplicons, and code are available as Supplemental Files. Raw Illumina FASTQ reads and associated metadata are available at NCBI: PRJNA608977.

Funding

This work was supported by the U.S. Geological Survey, Fort Collins Science Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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