Initial sample processing can influence the soil microbial metabarcoding surveys, revealed by Leucocalocybe mongolica fairy ring ecosystem

Figures & data

Table 1. General metabarcoding sequencing results of the soil samples.

Sample ID	16S rDNA			ITS
	Tags	N90 (bp)	OTUs	Tags	N90 (bp)	OTUs
LN-1	105391	441	7721	183533	352	1271
LN-2	103243	441	7528	185974	351	1181
LN-3	94364	441	7417	179043	349	1221
LN-4	96054	441	7297	178074	351	1211
LN-5	98194	441	7456	200309	350	1161
LN-6	108598	441	7744	172559	351	1091
DI-1	99046	441	7992	248485	352	1325
DI-2	104394	441	7927	189108	352	1265
DI-3	102727	441	7942	193608	352	1193
DI-4	108138	441	7705	188333	349	1123
DI-5	99954	441	7723	168489	351	1212
DI-6	104372	441	7452	203033	351	1185
DRY-1	104807	441	6654	243722	340	1121
DRY-2	106470	441	6352	210081	340	1144
DRY-3	109328	441	6357	191669	342	1094
DRY-4	99839	441	6501	187538	344	1054
DRY-5	107343	441	5932	183210	340	1052
DRY-6	107876	441	5386	183442	340	1009
MEAN	103341	441	7171	193900	347	1161

Note: S = Sobs, C = Chao1, A = Ace, SH = Shannon, SI = Simpson, GC = Good’s coverage.

Figure 1. Differences in preservation methods and sequencing results of soil samples across the three groups. (a) The timeline of sample processing. (b) The Venn diagram of OTUs from metabarcoding sequencing, in which 16S rDNA represents bacteria and ITS represents fungi. (c) The rare faction curves of the 18 samples from 16S rDNA and ITS metabarcoding, in which the x-axis represents sequence volume and the y-axis represents OTU numbers.

Figure 2. OTUs taxonomic analysis of the soil samples across the three groups. (a–d) Taxonomy of bacteria (a,c) and fungi (b,d) at phylum (a,b) and family (c,d) levels, in which the x-axis represents the sample identification number (ID) and the y-axis represents the relative abundance. (e,f) Heatmap of the top 10 families of bacteria (e) and fungi (f), in which the x-axis represents the sample ID and the y-axis represents the family. (g,h) The Kruskal-Wallis test of the top 5 families of bacteria (g) and fungi (h), in which the x-axis represents the relative abundance and the y-axis represents the family. Tricholomataceae was excluded from the results of fungi.

Table 2. Alpha diversity indices for the soil samples.

Sample ID	Bacteria						Fungi
	S	C	A	SH	SI	GC	S	C	A	SH	SI	GC
LN-1	7721	10157	10231	10.55	0.996	0.97	1271	1765	1771	2.61	0.42	0.998
LN-2	7528	10008	9923	10.5	0.996	0.97	1181	1614	1589	2.69	0.43	0.998
LN-3	7417	9794	9951	10.45	0.995	0.96	1221	1694	1669	2.82	0.45	0.998
LN-4	7297	9516	9531	10.41	0.995	0.97	1211	1822	1718	2.76	0.44	0.998
LN-5	7456	9764	9970	10.42	0.995	0.97	1161	1522	1480	2.89	0.46	0.999
LN-6	7744	9881	9980	10.54	0.996	0.97	1091	1505	1463	2.84	0.46	0.998
LN-MEAN	7527	9853	9931	10.48	0.996	0.97	1189	1654	1615	2.77	0.44	0.998
DI-1	7992	10530	10498	10.72	0.997	0.97	1325	1771	1738	2.59	0.41	0.999
DI-2	7927	10308	10260	10.64	0.997	0.97	1265	1694	1688	2.48	0.4	0.998
DI-3	7942	10499	10537	10.7	0.997	0.97	1193	1614	1598	2.48	0.4	0.998
DI-4	7705	10033	10084	10.58	0.997	0.97	1123	1598	1598	2.93	0.48	0.998
DI-5	7723	10045	10211	10.66	0.997	0.97	1212	1652	1647	2.67	0.43	0.998
DI-6	7452	9561	9545	10.57	0.997	0.97	1185	1577	1624	2.62	0.42	0.998
DI-MEAN	7790	10163	10189	10.65	0.997	0.97	1217	1651	1649	2.63	0.42	0.998
DRY-1	6654	8834	8798	10.23	0.997	0.98	1121	1565	1590	3.6	0.61	0.999
DRY-2	6352	8509	8354	9.95	0.995	0.98	1144	1672	1755	3.6	0.61	0.998
DRY-3	6357	8341	8214	9.96	0.994	0.98	1094	1522	1594	3.35	0.57	0.998
DRY-4	6501	8472	8404	10.27	0.997	0.98	1054	1676	1643	3.25	0.56	0.998
DRY-5	5932	7828	7585	9.92	0.994	0.98	1052	1450	1524	3.83	0.65	0.998
DRY-6	5386	7024	6857	10.02	0.996	0.98	1009	1644	1669	3.55	0.6	0.998
DRY-MEAN	6197	8168	8035	10.06	0.996	0.98	1079	1588	1629	3.53	0.60	0.998

Figure 3. Inter-group analysis of alpha diversity. The x-axis represents the indices of alpha diversity and the y-axis represents the groups of soil samples.

Figure 4. Beta diversity analysis of the soil samples across the three groups. (a,b) Heat map of weighted unifrac distance index based on UPGMA clustering from bacteria (a) and fungi (b), in which the bottom x-axis and right y-axis represent the sample ID, and the upper x-axis and left y-axis represent UPGMA clustering results. (c,d) The NMDS plot of bacteria (c) and fungi (d), in which MDS1 represents abundance distance matrix and MDS2 represents evolutionary distance matrix. (e,f) The map of Anosim analysis, in which the x-axis represents the group ID, the y-axis represents the relative difference grade, the R-value represents the difference level, and the P-value represents the significance of the difference.

Data availability statement

The raw amplicon sequencing dataset is available in the NCBI Sequence Read Archive under BioProject ID: PRJNA635768.