Algal environmental DNA data for 12 rivers and streams in the Milwaukee Metropolitan Sewerage District Planning Area, 2022-2024
Field methods: Water was taken from the surface layer at the center of the stream where flow was the highest. Water was drawn into a 60 milliliter (mL) syringe which was rinsed three times with stream water. A 25-millimeter, 1.0-micrometer pore-size Whatman nylon filter was then attached and primed with 5-10 mL of stream water before being removed. The syringe was refilled with 60 mL of stream water, the filter was reattached, and the water was filtered and discarded. This process was repeated until the filter became clogged. A Triton TE buffer preservative was injected into the filter followed by 50mL of air. The filter was capped, sample information recorded, and sent to Jonah Ventures. 23S sequencing laboratory methods: Sample Process: Sample barcodes were recorded and assigned a well within the 96 well plate or numbered extraction tube. A customized one-ton arbor press along with a removable leather punch was used to open the plastic casing of each filter. Once plastic casing was cut, sample barcodes were recorded and assigned a well within the 96 well plate or numbered extraction tube. The whole filter was removed and transferred to the extraction plate/tube using sterilized tweezers inside a laminar flow hood. The removable leather punch was sterilized between each eDNA filter. Plates or tubes were immediately processed or stored in -20 degrees C until the extraction process could be performed. Extraction: Genomic DNA from samples was extracted using the DNeasy PowerLyzer PowerSoil Kit (Cat# 12855-100) according to the manufacturers protocol. Genomic DNA was eluted into 100l and frozen at -20 degrees C. PCR: Forward primer: GGACAGAAAGACCCTATGAA Reverse primer: TGAGTGACGGCCTTTCCACT Primer notes: Algal Primer reference: Sherwood, Alison R., and Gernot G. Presting. 2007. Universal Primers Amplify a 23S rDNA Plastid Marker in Eukaryotic Algae and Cyanobacteria. Journal of Phycology 43, no. 3: 6058. https://doi.org/10.1111/j.1529-8817.2007.00341.x.; Hamsher, Sarah E., Katharine M. Evans, David G. Mann, Aloisie Poulkov, and Gary W. Saunders. 2011. Barcoding Diatoms: Exploring Alternatives to COI-5P. Protist 162, no. 3: 40522. https://doi.org/10.1016/j.protis.2010.09.005.; Cannon, M. V., J. Hester, A. Shalkhauser, E. R. Chan, K. Logue, S. T. Small, and D. Serre. 2016. In Silico Assessment of Primers for eDNA Studies Using PrimerTree and Application to Characterize the Biodiversity Surrounding the Cuyahoga River. Scientific Reports 6, no. 1: 22908. https://doi.org/10.1038/srep22908. A portion of the chloroplast trnL intron was PCR amplified from each genomic DNA sample using the p23SrV_f1 and Diam23Sr1 23S primers. Both forward and reverse primers also contained a 5 adaptor sequence to allow for subsequent indexing and Illumina sequencing. Each 40 L PCR reaction was mixed according to the Promega PCR Master Mix specifications (Promega catalog # M5133, Madison, WI) which included 12.5ul Master Mix, 0.5 M of each primer, 1.0 L of gDNA, and 10.5 L DNase/RNase-free H2O. DNA was PCR amplified using the following conditions: initial denaturation at 94 degrees C for 3 minutes, followed by 40 cycles of 30 seconds at 94 degrees C, 45 seconds at 55 degrees C, and 1 minute at 72 degrees C, and a final elongation at 72 degrees C for 10 minutes. Gel: To determine amplicon size and PCR efficiency, each reaction was visually inspected using a 2% agarose gel with 5 L of each sample as input. PCR Amplicon Cleanup: Amplicons were then cleaned by incubating amplicons with Exo1/SAP for 30 minutes at 37 degrees C following by inactivation at 95 degrees C for 5 minutes and stored at -20 degrees C. Barcoding PCR: A second round of PCR was performed to complete the sequencing library construct, appending with the final Illumina sequencing adapters and integrating a sample-specific,12-nucleotide index sequence. The indexing PCR included Promega Master mix, 0.5 M of each primer and 2 L of template DNA (cleaned amplicon from the first PCR reaction) and consisted of an initial denaturation of 95 degrees C for 3 minutes followed by 8 cycles of 95 degrees C for 30 sec, 55 degrees C for 30 seconds and 72 degrees C for 30 seconds. PCR Normal Pool: Final indexed amplicons from each sample were cleaned and normalized using SequalPrep Normalization Plates (Life Technologies, Carlsbad, CA). 25L of PCR amplicon is purified and normalize using the Life Technologies SequalPrep Normalization kit (cat#A10510-01) according to the manufacturers protocol. Samples are then pooled together by adding 5L of each normalized sample to the pool. Sequencing: Sample library pools were sent for sequencing on an Illumina NovaSeq 6000 (San Diego, CA) at the Texas A&M Agrilife Genomics and Bioinformatics Sequencing Core facility using the SP Reagent Kit v1.5 (500 cycles) (cat# 20028402). Necessary quality control measures were performed at the sequencing center prior to sequencing. Bioinformatics: Raw sequence data were demultiplexed using pheniqs v2.1.0 [1], enforcing strict matching of sample barcode indices (i.e, no errors). Cutadapt v3.4 [2] was then used remove gene primers from the forward and reverse reads, discarding any read pairs where one or both primers were not found at the expected location (5) with an error rate < 0.15. Read pairs were then merged using vsearch v2.15.2 [3], discarding resulting sequences with a length of < 300 bp, > 380 bp, or with a maximum expected error rate [4] > 0.5 bp. For each sample, reads were then clustered using the unoise3 denoising algorithm [5] as implemented in vsearch, using an alpha value of 5 and discarding unique raw sequences observed less than 8 times. Counts of the resulting exact sequence variants (ESVs) were then compiled and putative chimeras were removed using the uchime3 algorithm, as implemented in vsearch. For each final ESV, a consensus taxonomy was assigned using a custom best-hits algorithm and a reference database consisting of publicly available sequences (GenBank [6]) as well as Jonah Ventures voucher sequences records. Reference database searching used an exhaustive semi-global pairwise alignment with vsearch, and match quality was quantified using a custom, query-centric approach, where the % match ignores terminal gaps in the target sequence, but not the query sequence. The consensus taxonomy was then generated using either all 100% matching reference sequences or all reference sequences within 1% of the top match, accepting the reference taxonomy for any taxonomic level with > 90% agreement across the top hits. References: 1. Galanti, Shasha and Gunsalus (2021). Pheniqs 2.0: accurate, high-performance Bayesian decoding and confidence estimation for combinatorial barcode indexing. BMC Bioinformatics 22. 2. Martin (2011), Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal 17. 3. Torbjrn et al. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4 e2584 (2016). 4. Edgar and Flyvbjerg (2015), Error filtering, pair assembly and error correction for next-generation sequencing reads. Bioinformatics 31. 5. Edgar (2016), UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing. doi: https://doi.org/10.1101/081257. 6. Benson et al. (2005), GenBank. Nucleic Acids Res 33.