2.2- DNA Extraction, Amplification, and Sequencing
We processed each soil sample in the eDNA processing center of the UCSC Paleogenomics Lab following strict protocols to avoid contamination. Researchers working in the eDNA lab, which is isolated from PCR amplification products, wear sterile suits, face masks, hair nets, and gloves so as to minimize the possibility of contamination. Prior to DNA extraction, we homogenized the soil samples in the 50mL falcon tube in which they were collected so as to break up any larger pieces of sediment. Next, we removed any identifiable plant matter (leaves and roots) with sterile forceps. We then subsampled 0.25g aliquots of sediment for DNA extraction. We extracted DNA in duplicate for each of the six soil samples using the Qiagen PowerSoil kit and protocol (Qiagen, Germantown MD, USA), following the manufacturer’s protocol except for the final elution step, where we substituted the C6 elution buffer with Tris-EDTA-Tween buffer. We included one negative extraction control without soil.
We selected the ITS gene in plants (PITS) and fungi (FITS) as universal barcodes for metabarcoding. For PITS, we used primers described by Yao et al. 2010 (ITS-S2F - ATGCGATACTTGGTGTGAAT and ITS-S3R - GACGCTTCTCCAGACTACAAT) and for FITS, we used primers from White et al. 1990 (ITS5- forward - GGAAGTAAAAGTCGTAACAAGG) and Epp et al. 2012 (5.8S_fungi - reverse - CAAGAGATCCGTTGTTGAAAGTT). The expected amplicon length was 450 base pairs (bp) for PITS and 300 bp for FITS.
We used quantitative PCR (qPCR) to assess PCR inhibition in each extract and determine the appropriate number of PCR cycles for metabarcoding (as recommended in Murray, Coghlan, & Bunce, 2015). We performed qPCR with the Qiagen Multiplex PCR Master Mix and spiked SYBR Green 1 Dye (12.5uL Qiagen MM, 2uL of each 2uM primer, 0.6uL 1:2000 dilution SYBR Green 1 Dye, 5.9uL water, and 2uL extracted DNA). The Qiagen Multiplex PCR Master Mix introduces the least GC amplification bias of tested polymerases (Nichols et al. 2017). We amplified each extract in triplicate with PITS and FITS primers. For each replicate, we set up a serial dilution of 1:0, 1:1, and 1:3 extract to water proportions of the 2uL DNA extract input, and compared the qPCR Ct values across the dilution series. We observed no inhibition in any of the eDNA extracts, and decided to proceed with undiluted extracts. To avoid overamplification, we determined the optimal number of PCR cycles for each extract and PCR primer as the cycle after which the exponential amplification phase ended. We amplified extracts with PITS for 26-31 cycles and with FITS for 18-21 cycles.
We followed a ‘2-step’ protocol to build amplicon sequencing libraries (Nichols et al. 2017). The same reagent set up was used for metabarcoding PCR as for qPCR, but with the appropriate number of cycles and without SYBR Green 1 dye. The amplification primers included 5’ overhang Illumina (Illumina, San Diego, CA, USA) TruSeq adapter sequences, which allowed us to perform the indexing PCR directly after metabarcoding. For each of the six extracts we performed 24 replicate PCRs with PITS and 24 PCR replicates with FITS. We amplified four PITS and four FITS PCR replicates from the extraction negative control (no sediment) and added two additional PCR negative controls (no extract) for each primer.
Following metabarcoding PCR, we purified amplicon pools with SPRI beads (Beckman, Indianapolis, IN, USA). Next, we indexed all PCR products individually using Kapa Hifi (Roche, Pleasanton, CA, USA) to add eight base-pair dual indices and Illumina sequencing adapters to our amplicon pools (12.5uL Kapa Hifi, 5.5uL water, 1uL of each 10uM forward and reverse index, and 5uL purified PCR product), followed by a second SPRI bead clean. We used unique combinations of dual indices for each PCR replicate, though each individual index was used multiple times across different amplicon pools. We then quantified the concentration of DNA in the purified amplicon pools with a Nanodrop (Thermo Scientific, Waltham, MA, USA). We used these estimates of DNA concentration to pool the PCR amplification products into equimolar ratios in two pools, one for PITS and one for FITS. We then quantified the two pools with a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA) and estimated average fragment sizes with a fragment analyzer.
To detect potential index swapping (the incorrect assignment of sequences to an index as a result of blurring of index signals between adjacent clusters, van der Valk et al., 2019) during sequencing, we amplified the PITS metabarcode from an extract of spiral ginger( Costus pulverulentus ), provided to us by Kathleen Kay’s lab at UCSC. The Kay lab collected samples from La Selva, Costa Rica under permit R-056-2019-OT-CONAGEBIO and extracted floral C. pulverulentus tissue with the Qiagen plant mini kit. Spiral ginger is native to the neotropics and not found in either California or Alaska. We generated three replicate PCR amplicon libraries from the spiral ginger extract following the 2-step protocol described above. Following sequencing and demultiplexing, we estimated the rate of index hopping based on the number of non-ginger sequences assigned to the ginger dual indices.
We pooled and sequenced 308 sediment and three spiral ginger libraries (a total of 311 libraries) on an Illumina Miseq using v3 chemistry and a 2x300 approach. We targeted 100,000 reads per FITS library and 50,000 reads per PITS library, based on the anticipated higher taxonomic diversity amplified by FITS and the anticipated higher discard rate of FITS-amplified sequences due to the incompleteness of the fungal taxonomy databases.