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.