利用lmperm包进行置换检验

1准备细菌相对丰度文件

2读入excel文件,以属水平数据为例

library(readxl)
bac.g <- read_excel(file.choose(),sheet = 1)

3选择数据子集

bac.g.b <- subset(bac.g,bac.g$b=="1")
fix(bac.g.b) #设定goat为字符型变量
bac.g.b<-as.data.frame(bac.g.b) 

4置换次数设定为Prob，结果保存至bac.g.b.results文件中，文件共有135列数据、因变量从第9列开始，循环从9-135

i<-9
sink("bac.g.b.results",append=TRUE)
while(i<=135){ 
  print(
      summary(aovp(bac.g.b[,i]~goat+beater,bac.g.b,perm="Prob"))
    );i<-i+1
}
sink()

5准备excel文件放置整理后的统计结果，第一页为菌名，属水平一共127个菌

	bac_g	bac_f	bac_o	bac_c	bac_p
1	Prevotella_1	0319-6G20	Absconditabacteriales_	Alphaproteobacteria	Bacteria_unclassified
2	F082_ge	Absconditabacteriales__fa	Aeromonadales	Anaerolineae	Bacteroidetes
3	Rikenellaceae_RC9_gut_group	Acidaminococcaceae	Alphaproteobacteria_unclassified	Bacteria_unclassified	Chloroflexi
4	Treponema_2	Alphaproteobacteria_unclassified	Alteromonadales	Bacteroidia	Cyanobacteria
5	WCHB1-41_ge	Anaerolineaceae	Anaerolineales	Clostridia	Elusimicrobia
6	Prevotellaceae_UCG-003	Anaeroplasmataceae	Anaeroplasmatales	Deltaproteobacteria	Euryarchaeota
7	Butyrivibrio_2	Bacteria_unclassified	Bacteria_unclassified	Elusimicrobia	Fibrobacteres
8	Bacteroidales_BS11_gut_group_ge	Bacteroidales_BS11_gut_group	Bacteroidales	Endomicrobia	Firmicutes
9	Ruminobacter	Bacteroidales_RF16_group	Bacteroidia_unclassified	Erysipelotrichia	Kiritimatiellaeota
10	Fibrobacter	Bacteroidales_UCG-001	Clostridiales	Fibrobacteria	Lentisphaerae
11	Christensenellaceae_R-7_group	Bacteroidales_unclassified	Deltaproteobacteria_unclassified	Gammaproteobacteria	Patescibacteria
12	Erysipelotrichaceae_UCG-004	Bacteroidetes_BD2-2	Desulfovibrionales	Gracilibacteria	Planctomycetes
13	Lachnospiraceae_XPB1014_group	Bacteroidia_unclassified	Desulfuromonadales	Kiritimatiellae	Proteobacteria
14	Bacteroidales_RF16_group_ge	Christensenellaceae	Elusimicrobiales	Lentisphaeria	Spirochaetes
15	Bacteroidales_UCG-001_ge	Clostridiales_unclassified	EMP-G18	Melainabacteria	Tenericutes
16	Muribaculaceae_ge	Clostridiales_vadinBB60_group	Endomicrobiales	Methanobacteria	Verrucomicrobia
17	Mollicutes_RF39_ge	COB_P4-1_termite_group	Enterobacteriales	Methanomicrobia
18	Ruminococcaceae_NK4A214_group	Defluviitaleaceae	Erysipelotrichales	Mollicutes
19	uncultured	Deltaproteobacteria_unclassified	Fibrobacterales	MVP-15
20	Saccharofermentans	Desulfovibrionaceae	Gastranaerophilales	Negativicutes
21	Ruminococcus_1	Desulfuromonadaceae	Izimaplasmatales	Oligosphaeria
22	Ruminococcaceae_UCG-005	Desulfuromonadales_unclassified	LD1-PB3	Planctomycetacia
23	Prevotellaceae_unclassified	Elusimicrobiaceae	Methanobacteriales	Proteobacteria_unclassified
24	Succiniclasticum	EMP-G18_fa	Methanomassiliicoccales	Saccharimonadia
25	Prevotellaceae_UCG-001	Endomicrobiaceae	Methanomicrobiales	Spirochaetia
26	Lachnospiraceae_unclassified	Enterobacteriaceae	Mollicutes_RF39	Thermoplasmata
27	Ruminococcaceae_UCG-014	Erysipelotrichaceae	MVP-15_or	Verrucomicrobiae
28	Clostridiales_unclassified	F082	Mycoplasmatales
29	Ruminococcaceae_UCG-002	Family_XIII	Myxococcales
30	Ruminococcaceae_UCG-010	Fibrobacteraceae	Oceanospirillales
31	Gastranaerophilales_ge	Gastranaerophilales_fa	Oligoflexales
32	Lachnospiraceae_AC2044_group	Halomonadaceae	Oligosphaerales
33	uncultured_ge	Idiomarinaceae	Pedosphaerales
34	vadinBE97_ge	Izimaplasmatales_fa	Pirellulales
35	Succinivibrionaceae_UCG-002	Lachnospiraceae	Proteobacteria_unclassified
36	Papillibacter	LD1-PB3_fa	Rhodospirillales
37	Ruminococcaceae_ge	Marinifilaceae	Saccharimonadales
38	Flexilinea	Marinilabiliaceae	Selenomonadales
39	Clostridiales_vadinBB60_group_ge	Methanobacteriaceae	Spirochaetales
40	horsej-a03	Methanomethylophilaceae	Victivallales
41	Lachnospiraceae_NK4A136_group	Methanomicrobiaceae	WCHB1-41
42	MVP-15_ge	Mollicutes_RF39_fa
43	Ruminococcaceae_unclassified	Muribaculaceae
44	CAG-352	MVP-15_fa
45	Anaerosporobacter	Mycoplasmataceae
46	p-251-o5_ge	Myxococcales_unclassified
47	Bacteria_unclassified	Oligosphaeraceae
48	Ruminococcaceae_UCG-013	p-251-o5
49	Pseudobutyrivibrio	Pedosphaeraceae
50	Ruminococcus_2	PeH15
51	Lachnoclostridium_10	Peptococcaceae
52	Pirellula	Pirellulaceae
53	Victivallaceae_ge	Prevotellaceae
54	Ruminococcaceae_V9D2013_group	Proteobacteria_unclassified
55	Lachnospiraceae_NK3A20_group	Rhodospirillales_unclassified
56	Prevotellaceae_UCG-004	Rikenellaceae
57	Prevotellaceae_NK3B31_group	Ruminococcaceae
58	Ruminiclostridium_6	Saccharimonadaceae
59	probable_genus_10	Spirochaetaceae
60	Bacteroidales_unclassified	Succinivibrionaceae
61	Anaerovorax	Terasakiellaceae
62	Lachnospiraceae_ge	uncultured
63	Anaeroplasma	vadinBE97
64	Absconditabacteriales__ge	Veillonellaceae
65	Rhodospirillales_unclassified	Victivallaceae
66	Mycoplasma	Victivallales_unclassified
67	LD1-PB3_ge	WCHB1-41_fa
68	Lachnospiraceae_FCS020_group
69	Methanobrevibacter
70	Candidatus_Saccharimonas
71	Lachnospiraceae_UCG-008
72	Bacteroidia_unclassified
73	Lachnospiraceae_ND3007_group
74	Ruminococcaceae_UCG-012
75	Elusimicrobium
76	Veillonellaceae_UCG-001
77	CPla-4_termite_group
78	Methanomicrobium
79	Bacteroidetes_BD2-2_ge
80	Sediminispirochaeta
81	Izimaplasmatales_ge
82	Z20
83	Desulfovibrio
84	p-1088-a5_gut_group
85	Marinospirillum
86	U29-B03
87	PeH15_ge
88	Ruminococcaceae_UCG-004
89	Prevotellaceae_YAB2003_group
90	Coprococcus_2
91	Moryella
92	Lachnospiraceae_UCG-009
93	Lachnospiraceae_UCG-006
94	Alphaproteobacteria_unclassified
95	Terasakiellaceae_unclassified
96	Desulfuromonas
97	Deltaproteobacteria_unclassified
98	XBB1006
99	Christensenellaceae_unclassified
100	Sphaerochaeta
101	Mailhella
102	COB_P4-1_termite_group_ge
103	Pedosphaeraceae_ge
104	Oribacterium
105	Anaerovibrio
106	Succinimonas
107	Lachnospiraceae_UCG-010
108	Candidatus_Endomicrobium
109	Proteobacteria_unclassified
110	Desulfuromonadales_unclassified
111	Aliidiomarina
112	EMP-G18_ge
113	Flavonifractor
114	Ruminococcaceae_UCG-009
115	Klebsiella
116	Myxococcales_unclassified
117	Ruminiclostridium_5
118	Spirochaetaceae_unclassified
119	Methanosphaera
120	0319-6G20_ge
121	Ruminococcaceae_UCG-007
122	Defluviitaleaceae_UCG-011
123	Victivallaceae_unclassified
124	Victivallales_unclassified
125	possible_genus_Sk018
126	Erysipelotrichaceae_unclassified
127	Methanobacteriaceae_unclassified

第二页放置文件，先将各组平均值准备好

microbe	taxonomy	item	YM+SB	YM+BB	YM-	QIA+BB	QIA-
bac	genus	Prevotella_1	23.30842444	23.07721286	22.42106592	26.01562212	19.55163517
bac	genus	F082_ge	20.80524039	18.59373306	18.86473074	18.18234246	24.68416661
bac	genus	Rikenellaceae_RC9_gut_group	8.394831813	7.301305947	7.777839723	7.69692754	8.89710732
bac	genus	Treponema_2	2.879574177	3.320699141	3.094990767	5.066247403	4.148058497
bac	genus	WCHB1-41_ge	2.839534463	2.441781585	3.388955613	4.41439074	3.64009815
bac	genus	Prevotellaceae_UCG-003	2.545339266	2.982857002	2.403730984	3.417546927	2.341680934
bac	genus	Butyrivibrio_2	2.179614133	1.879141151	2.451410829	2.126128419	2.913995311
bac	genus	Bacteroidales_BS11_gut_group_ge	2.40655748	2.436610503	2.531583068	1.762313972	2.032382983
bac	genus	Ruminobacter	1.885639618	2.158922457	2.579704941	1.899031917	2.085402369

 6用execl打开结果文件，选中第一列，数据→分列、空格为分隔符

筛选，选择残差项，复制出均方值到整理表中，（注意：在第4列，不要看表头），均方值开二次方也就是SEM值；

筛选beater和goat项，复制出P值项和项目项到新页中（注意：第5列，不要看表头！）整理为如下样式

Item	P-value	s
goat1	0.1542	1
beater1	0.902	1
goat1	0.06745	2
beater1	0.39355	2
goat1	0.33	3
beater1	0.3021	3
goat1	0.2176	4

插入数据透视表，值-P值；行-s；列-item

平均值项:P-value	Item
s	beater1	goat1
1	0.902	0.1542
2	0.39355	0.06745
3	0.3021	0.33
4	0.6667	0.2176
5	0.6667	0.9855
6	0.7255	0.1542
7	0.36571	0.05915
8	0.9608	0.1545

复制P值结果到整理表中，最终结果如下表所示（本例中仅显示P-value2的整理过程）

			Protocol						P-value1					P-value2
microbe	taxonomy	item	YM+SB	YM+BB	YM-	QIA+BB	QIA-	SEM1	beadbeating1	goat1	protocol1	protocol1:bead	SEM2	beater1	goat1
bac	genus	Prevotella_1	23.30842444	23.07721286	22.42106592	26.01562212	19.55163517	54.711	1	0.8235	0.2024	0.5104	4.278083683	0.902	0.1542
bac	genus	F082_ge	20.80524039	18.59373306	18.86473074	18.18234246	24.68416661	23.822	0.2681	0.3017	0.0018	0.2468	2.279473623	0.39355	0.06745
bac	genus	Rikenellaceae_RC9_gut_group	8.394831813	7.301305947	7.777839723	7.69692754	8.89710732	4.3457	0.5641	0.8824	0.0752	0.4951	1.376117728	0.3021	0.33
bac	genus	Treponema_2	2.879574177	3.320699141	3.094990767	5.066247403	4.148058497	4.2476	0.3591	0.7059	0.5158	0.7647	1.18046601	0.6667	0.2176
bac	genus	WCHB1-41_ge	2.839534463	2.441781585	3.388955613	4.41439074	3.64009815	2.9505	0.3706	0.4815	0.2094	0.9804	0.878510102	0.6667	0.9855
bac	genus	Prevotellaceae_UCG-003	2.545339266	2.982857002	2.403730984	3.417546927	2.341680934	2.23211	0.902	0.6863	0.7477	0.3851	0.949315543	0.7255	0.1542
bac	genus	Butyrivibrio_2	2.179614133	1.879141151	2.451410829	2.126128419	2.913995311	0.51451	0.4655	0.6667	0.2908	0.1834	0.254715528	0.36571	0.05915
bac	genus	Bacteroidales_BS11_gut_group_ge	2.40655748	2.436610503	2.531583068	1.762313972	2.032382983	0.6799	0.2182	0.9412	0.009	0.902	0.747596148	0.9608	0.1545