Synthetic spikes were designed with three key elements: (i) primer binding sites (PBSs) from the common genes used for identification of prokaryotes (P), eukaryotes (E) and fungi (F), respectively; (ii) an optimised synthetic stuffer sequence of the same length and GC content as the in vivo target and (iii) a readily available, easy to handle source of synthetic spike DNA (Fig. 1).

Synthetic spike design. P, E and F synthetic spikes were designed using PBS sequences, together with the length and GC content of amplicons from prokaryotes (P), eukaryotes (E) and fungi (F), respectively. For P synthetic spikes the primer binding sites (PBS) shown in orange, for E in green and for F in blue

PBSs are based on the three sets of PCR primers chosen for microbiota amplification. For prokaryotes (P), the prokaryotic 16S rRNA V4 region primers 515F and 806R [19 (link)] were used as they are commonly used in soil studies. For eukaryotes (E), we used the 18S primer pair, F1427 and R1616 [20 (link)], that targets a broad range of eukaryotic taxa including algae, diatoms, animals, excavates (protists, flagellates), fungi and moulds. The third primer pair specifically targets fungi (F), ITS1F and ITS2R and are widely used in soil microbiota studies [21 (link)]. These primers target the variable sized ITS fragment of Ascomycota and Basidiomycota, common phyla in forest soils [22 (link)]. As variability in amplicon length can bias PCR amplification efficiency, the length of the stuffer sequences of P (16S rRNA) and E (18S rRNA) were matched to the length of natural PCR products. Fungal ITS amplicons show more variability in length, but based on the results of our previous sequencing, the most common size was 272 bp [23 (link)]; therefore, a stuffer sequence of 272 bp was used in designing the F synthetic spike (Fig. 1). In each case, GC content was designed to be similar to their environmental gene counterparts with sequences designed using a random DNA generator (https://www.faculty.ucr.edu). P, E and F synthetic spikes were synthesised by Geneart (Invitrogen) and supplied cloned in plasmid pMA-T, forming pSpike-P, pSpike-E and pSpike-F, respectively. Plasmid were transformed into Escherichia coli and deposited at https://www.addgene.com as plasmids #101172, #101173 and #101174.
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