A subset of our ultra-low biomass samples were also subjected to amplicon sequencing for direct comparison with the shotgun metagenomic sequencing approach. For these samples, the first stage PCR was performed with the extracted genomic DNA as a template and the ITS1F-ITS2R45 primers for fungi and 16S 341F-805R46 primers for bacteria. Details of these primer sequences can be found in Table Table2.2. KAPA HiFi HotStart master mix was used with a total reaction volume of 25 µL. For DNA input amount, 3 µL and 10 µL of DNA templates were used for fungi and bacteria, respectively. The cycling condition was 95 °C for 3 min, amplification cycles with 95 °C for 30 s, 65 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The fungal samples were amplified with 15 cycles and the bacteria samples were amplified with 25 cycles. The PCR products were then purified with AMPure XP beads (Beckman Coulter) before performing the second stage PCR.

The second stage PCR (Indexing PCR) was performed according to the recommendations in Illumina’s “16S Metagenomic Sequencing Library Preparation” application note. This step uses a limited cycle PCR to complete the Illumina sequencing adapters and add dual-index barcodes to the amplicon target. Five microliters of the intermediate PCR product from the first stage PCR (Amplicon PCR) were used as template for the indexing PCR and samples were amplified with eight PCR cycles. Nextera XT v2 indices were used for dual-index barcoding to allow pooling of the amplicon targets for sequencing.

Finished amplicon libraries were quantitated using Promega’s QuantiFluor dsDNA assay and the average library size was determined on an Agilent Tapestation 4200. Library concentrations were then normalised to 4 nM and validated by qPCR on a QuantStudio-3 real-time PCR system (Applied Biosystems), using the Kapa library quantification kit for Illumina platforms (Kapa Biosystems). The libraries were then pooled at equimolar concentrations and sequenced on the Illumina MiSeq platform with 20% PhiX spike-in and at a read-length of 300 bp paired-end (MiSeq V3 reagents).

After sequencing, raw reads were first trimmed from adapter sequences, low-quality bases and short reads using Cutadapt (v.1.8.1)43. After trimming, the R1 and R2 reads were first paired with minimum overlap of 10 bp and subsequently aligned against UNITE ITS database (v.7.1) for the ITS sequences and SILVA 16S database (release 132) for the 16S sequences using command line blastn36 (version 2.2.28 + ). Results from blastn alignments were also converted to read-match archive (rma) format for visualisation with the MEGAN5 software to facilitate direct comparison with the metagenomic sequencing analysis. The default LCA parameters were used.

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