Metabarcoding Data from an Inventory of Freshwater Invertebrates from the Miller Creek Watershed, Kenai Peninsula, Alaska, USA

Study Extent

The study extent included 3 D-net samples, 3 Ekman dredge samples, and 2 Wisconsin net samples in Vogel Lake; 2 D-net samples, 2 Ekman dredge samples, and 1 Wisconsin net sample in North Vogel Lake; and 3 D-net samples in Miller Creek, a total of 16 invertebrate samples per visit, sampled twice per year for a total of 32 samples per year.

Sampling

Field methods generally followed the methods of Massengill (2014, 2017). We took vertical plankton tows in the deepest parts of the lakes using an Aquatic Research Instruments Wisconsin net. We sampled littoral areas using with D-nets. We obtained benthic samples using either an AMS Incorporated model 445.11 Ekman dredge or an AMS Incorporated model 445.60 stainless steel dredge. Most benthic samples were sorted using a series of sieves.

Method steps

1. Metabarcoding samples were stored in a -23 °C freezer exept when samples were being processed. Invertebrates were separated from debris by hand under a dissecting microscope. Care was taken to reduce possible crosscontamination of DNA among samples. Samples were shipped out on ice on September 29, 2021, arriving the next day at MR DNA (Shallowater, Texas, http://www. mrdnalab.com). We chose to use the mlCOIintF/jgHCO2198 primer set of Leray et al. (2013) for PCR, targeting a 313 bp region of the COI DNA barcoding region. The mlCOIintF/jgHCO2198 primer pair was used with barcode on the forward primer in a 30–35 PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 °C for 3 minutes, followed by 30– 35 cycles of 94 °C for 30 s, 53 °C for 40 seconds and 72 °C for 1 minute, after which a final elongation step at 72 °C for 5 minutes was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare an illumina DNA library. Sequencing was performed at MR DNA on a MiSeq following the manufacturer’s guidelines. We processed the raw sequence data on the USGS Yeti supercomputer (USGS Advanced Research Computing, 2021) using R, version 4.1.1 for manipulating data and Je, version 2.0.RC (Girardot et al., 2016) for demultiplexing. The raw data included reads in alternating directions with the sample barcodes only on one read. Accordingly, we exectued the je demultiplex command, accepting the defaults that only require one of the two reads to contain a sample barcode. Raw read data were processed using MetaWorks, version 1.9.5 (Porter and Hajibabaei, 2020), running the default analysis options for metazoan COI DNA barcode sequences. Sequences were identified using the RDP classifier, version 2.13 (Wang et al., 2007) and the CO1 Classifier, version 4.0.1 reference library (Porter, 2017; Porter and Hajibabaei, 2018). Finally, we also compared the sequences to libraries we had obtained in previous metabarcoding projects on the Kenai Peninsula.

Annelida, Arthropoda, Mollusca

1. Annelida
   rank: Phylum
2. Arthropoda
   rank: Phylum
3. Mollusca
   rank: Phylum

The geographic coverage is part of the Miller Creek watershed including North Vogel Lake, Vogel Lake, and Miller Creek.