DNA Sequences

During observational sampling bouts of certain organisms, NEON field ecologists select a subset of aquatic and terrestrial organisms for generation of DNA sequence data and metadata. Many of the samples are also archived into the NEON biorepository. DNA analysis serves a number of purposes, including verification of taxonomy of specimens that do not receive expert identification, clarification of the taxonomy of rare or cryptic species, characterization of diversity using molecular markers, and functional gene characterization. These data are open access and discoverable through the NEON data portal.

DNA sequences of organisms vary by site type and include:

  • Benthic, surface water, and soil microbes
  • Macroinvertebrates and zooplankton
  • Fish, ground beetles, mosquitoes, and small mammals

Shotgun metagenome sequences

The following data products are created from collected samples that are frozen on dry ice and shipped to an analytical facility for DNA extraction, sample preparation, shotgun metagenomic sequencing and high-throughput sequence analysis using primer sets targeting the small subunit of the ribosomal RNA gene.  

Benthic microbe marker genes and metagenomes

Site Type: Streams

Benthic microbe samples are collected at the same time and location as periphyton (microalgae) samples three times per year in wadeable streams during aquatic biology bout windows, roughly in spring, summer, and fall. Samples are scrubbed from rock surfaces (epilithon) and filtered, or collected from the sediments (epipsammon, epipelon) or plant surface (epiphyton). A duplicate set of filtered samples collected from rock scrubs are sent to the archive facility.

Surface water microbe marker genes and metagenomes

Site Type: Streams, Rivers, Lakes

Surface water microbe samples are collected at the same time and location as surface water cell count samples and surface water chemistry samples once per month in wadeable streams (12 times per year) and every-other month in lakes and rivers (6 times per year). In wadeable streams, surface water microbe samples are collected near the downstream surface water sensor location. In lakes and rivers, microbial samples are collected near the the buoy-mounted sensor station, where sampling depth is dependent on stratification. At flow-through lakes, surface water microbes are also collected near the inlet, and outlet sensors. A duplicate set of samples are sent directly to the archive facility.

Soil microbe marker genes and metagenomes

Site Type: Terrestrial

Soil sampling occurs annually during the historic peak in vegetation greenness at three randomly assigned base plots are terrestrial sites. Soil samples are collected to a maximum depth of 30 cm, with organic and mineral soils sampled separately. Subsamples of homogenized soil (rocks, roots and organic debris removed) from each of the 3 sampling locations are combined to form a plot-level composite sample that is used for metagenomics analysis.

Metabarcodes

The following data products are created from samples that are shipped to an external facility for homogenization and high-throughput sequencing (metabarcoding) targeting two regions of the COI gene.

Macroinvertebrate DNA metabarcodes

Site Type: Streams, Rivers, Lakes

Benthic macroinvertebrate DNA samples are collected three times per year at wadeable stream, river, and lake sites during aquatic biology bout windows, roughly in spring, summer, and fall, at the same time and location as morphological taxonomy samples. Samples from the spring and fall bouts are shipped directly to the archive. Macroinvertebrate samples are collected using the most appropriate sampler for the habitat type, including Surber, Hess, hand corer, modified kicknet, and D-frame sweep. In wadeable streams, samples are collected in the most dominant habitat type (e.g. riffles, runs, pools, step pools). In lakes and rivers, samples are collected in littoral areas. Samples are preserved in ethanol in the field and returned to the domain support facility for a preservative change prior to shipping to an external facility. Samples from the mid-summer bout are used for metabarcoding.

Zooplankton DNA metabarcodes

Site Type: Lakes

Zooplankton DNA samples are collected three times per year at lake sites during aquatic biology bout windows, roughly in spring, summer, and fall. Samples from the mid-summer bout are used for metabarcoding. Samples from the spring and fall bouts are shipped directly to the archive. Samples are collected using either a tow net (water deeper than 4 m) or a Schindler-Patalas trap (water shallower than 4 m) depending on the depth at the sampling location. Zooplankton samples are collected near the buoy-mounted sensor station, as well as the inlet and outlet sensor sets. Samples are preserved in ethanol in the field and returned to the domain support facility for a preservative change prior to shipping to an external facility.

DNA barcode sequences

The following data products are created from tissue samples that are shipped to an external lab for DNA extraction. Target sequences are amplified via PCR. Barcodes of cytochrome oxidase I are generated per specimen.

Fish DNA barcodes

Site Type: Streams, Lakes

Fish sampling is conducted twice a year at most wadeable streams and lakes, roughly in the fall and spring. During each sampling bout, fin clips are taken from up to 5 specimens per species to be used for DNA barcoding. The primary objective for DNA barcoding fish is to elucidate cryptic and hybrid species as well as for taxonomic verification.

Ground beetle DNA barcodes

Site Type: Terrestrial

At each terrestrial site, ground beetles samples are collected bi-weekly throughout the growing season using pitfall traps. During these sampling bouts, a subset of beetle legs are collected from up to 10 individuals per species per site. Up to 95 of these samples per site per year are used for DNA barcoding. Beetles that are rare, particularly difficult to identify, or poorly represented in previous collection events are prioritized for DNA sequencing. Only beetle specimens that have been identified by an expert taxonomist are eligible for DNA barcoding.

Mosquito DNA barcodes

Site Type: Terrestrial

At each terrestrial site, samples are collected periodically throughout the active mosquito season using CO2 traps. During these sampling bouts, a subset of mosquito legs are collected from up to 10 individuals per species per NEON domain. Up to 95 of these samples per domain per year are used for DNA barcoding.

Small mammal DNA barcodes

Site Type: Terrestrial

At terrestrial field sites, live trapping is conducted 4-6 times per year to collect small mammal observations. During these sampling bouts, up to 240 ear punches are collected from live specimens at each site per year. Up to 95 of these samples per domain per year are used for DNA barcoding.

All data are openly available through the NEON data portal.

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