高通量测序技术分析无特定病原体级实验小鼠肠道的菌群组成

doi:10.16150/j.1671-2870.2020.01.012

摘要/Abstract

摘要：

目的：应用高通量测序技术检测无特定病原体（specific pathogen free, SPF）级实验小鼠肠道的菌群组成,分析其细菌携带状况。方法：应用高通量测序技术,对14个品系共65只SPF级实验小鼠的回盲部肠道内容物样本中细菌16S rRNA V3~V4区进行深度测序,分析样本的菌群组成、丰度和Alpha多样性,并在属水平上筛选葡萄球菌属、假单胞菌属、支原体和棒状杆菌属。结果：65只SPF级健康成年小鼠的肠道内容物样本经数据过滤和质控后,共得到144 318条高质量序列用于后续分析,结果提示相同品系样品间菌群丰度表现出明显的差异。所有样本的序列主要集中在以下8个门,依次为厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）、变形菌门（Proteobacteria）、疣微菌门（Verrucomicrobia）、放线菌门（Actinobacteria）、Saccharibacteria、软壁菌门（Tenericutes）和螺旋菌门（Spirochaetae）。在属水平上检测到了葡萄球菌属、假单胞菌属、支原体和棒状杆菌属。结论：相同品系的SPF级实验小鼠肠道菌群在物种组成、丰度及Alpha 多样性方面也会表现出显著差异,有必要致力于研究各种方法以使实验动物的肠道菌群在可控和可监测范围内,高通量测序技术提供了这一可能。

关键词: 无特定病原体级实验小鼠, 肠道菌群, 16S rRNA, 高通量测序

Abstract:

Objective: To analyze the intestinal microbial composition in specific pathogen free(SPF) mice using high-throughput sequencing. Methods: A total of 65 SPF mice of 14 strains were collected. Samples of fresh ileocecus content from the SPF mice were examined using high-throughput sequencing, and 16S rRNA V3-V4 variable regions of bacterium were deeply sequenced. The composition, alpha diversity, and the abundance of microbial flora in ileocecus content were explored, while Staphylococcus, Pseudomonas, Mycoplasma and Corynebacterium were identified at the genus level. Results: Altogether 144 318 high-quality genome sequences were obtained after data filter and quality control. Remarkable differences were found in Alpha diversity (species richness, diversity and evenness) of the microbial composition between samples from mice of the same strains. At the phylum level, the main sequences were largely concentrated as follows: Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Actinobacteria, Saccharibacteria, Tenericutes and Spirochaetae. At the genus level, Staphylococcus, Pseudomonas, Anaeroplasma and Corynebacterium were detected in some samples. Conclusions: The composition, alpha diversity and the abundance of the microbial flora in ileocecus contents are substantially different in mice of same strain. There is a need to keep intestinal microbial composition in SPF mice under control, and high-throughput sequencing may offer such possibility.

Key words: Specific pathogen free mice, Intestinal microbial composition, 16S rRNA, High-throughput sequencing

中图分类号:

R446.139

李惠, 冯洁, 韩立中. 高通量测序技术分析无特定病原体级实验小鼠肠道的菌群组成[J]. 诊断学理论与实践, 2020, 19(1): 55-62.

LI Hui, FENG Jie, HAN Lizhong. Analysis of intestinal microbial composition in specific pathogen free mice using high-throughput sequencing[J]. Journal of Diagnostics Concepts & Practice, 2020, 19(1): 55-62.

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品系只数	样本编号
C57 11	1~8、10、54、55
BALB/c 11	9、18~20、41~46、47（BALB/c,mmdl）
Nu/Nu 3	11、37、38
Db 3	12、13、14
Ob 3	15、16、17
ICR 15	21、22、28、33~36、49~52、58~61
C57BL/6 11	23~27、30~32、39、40、65
EIIA-Cre 1	29
suid-beige 1	48
CBA 1	53
Fek-cre 1	56
FAH-Rage 1	57
NAV 1	62
Ai32 2	63~64

样本号	序列	Alpha 多样性
样本号	序列	OTU	Ace指数	Chao1指数	Coverage指数	Shannon指数	Simpson指数
1	25549	293	347(325,384)	344(319,393)	0.997 808	3.89(3.87,3.91)	0.043 4(0.042 3,0.044 4)
2	25549	238	293(269,338)	313(273,398)	0.998 121	4.24(4.23,4.26)	0.022 8(0.022 3,0.023 3)
3	25549	250	302(279,341)	323(286,398)	0.997 926	3.97(3.95,3.99)	0.038 2(0.037 3,0.039 1)
4	25549	229	285(261,326)	291(260,353)	0.997 886	3.48(3.46,3.50)	0.082 9(0.080 5,0.085 3)
5	25549	167	202(184,237)	202(182,251)	0.998 708	3.59(3.57,3.61)	0.051 4(0.050 3,0.052 5)
6	25549	192	277(249,319)	250(220,312)	0.998 043	2.71(2.69,2.73)	0.157 6(0.154 6,0.160 7)
7	25549	222	274(252,313)	281(252,340)	0.997 847	3.35(3.33,3.37)	0.071 3(0.069 8,0.072 8)
8	25549	212	265(242,305)	259(235,308)	0.998 082	3.26(3.24,3.28)	0.092 3(0.089 9,0.094 6)
10	25549	345	403(381,440)	426(389,494)	0.997 260	4.06(4.04,4.08)	0.038 9(0.037 9,0.039 8)
54	25549	342	424(394,473)	459(406,556)	0.996 986	4.21(4.20,4.23)	0.027 2(0.026 6,0.027 8)
55	25549	213	265(242,306)	276(243,344)	0.998 121	2.84(2.81,2.86)	0.172 2(0.168 1,0.176 3)

样本号	序列	Alpha 多样性
样本号	序列	OTU	Ace指数	Chao1指数	Coverage指数	Shannon指数	Simpson指数
9	25549	236	337(305,382)	293(265,347)	0.997 730	3.36(3.34,3.38)	0.071 0(0.069 6,0.072 5)
18	25549	315	367(346,403)	372(344,426)	0.997 730	3.99(3.97,4.01)	0.045 4(0.044 1,0.046 6)
19	25549	377	439(416,475)	439(412,488)	0.996 986	3.91(3.89,3.93)	0.052 7(0.051 3,0.054 2)
20	25549	474	535(513,569)	535(509,582)	0.996 947	4.82(4.80,4.84)	0.015 3(0.014 9,0.015 6)
41	25549	178	246(221,286)	245(206,336)	0.998 552	3.63(3.61,3.65)	0.051 2(0.049 9,0.052 5)
42	25549	164	182(172,204)	181(170,210)	0.999 139	3.56(3.54,3.57)	0.053 6(0.052 3,0.054 9)
43	25549	70	97(82,128)	78(72,101)	0.999 530	2.63(2.61,2.64)	0.107 7(0.106 1,0.109 3)
44	25549	95	157(132,199)	122(105,168)	0.999 021	2.92(2.91,2.93)	0.070 9(0.070 1,0.071 7)
45	25549	88	132(112,166)	122(99,196)	0.999 256	2.78(2.76,2.79)	0.116 9(0.114 2,0.119 5)
46	25549	81	92(85,115)	111(89,193)	0.999 452	2.84(2.82,2.85)	0.090 3(0.088 8,0.091 8)
47	25549	161	195(178,229)	199(177,251)	0.998 708	3.40(3.39,3.42)	0.058 1(0.057 0,0.059 2)

样本号	序列	Alpha 多样性
样本号	序列	OTU	Ace指数	Chao1指数	Coverage指数	Shannon指数	Simpson指数
21	25549	465	522(501,554)	524(498,571)	0.997 025	4.43(4.41,4.45)	0.037 9(0.036 8,0.039 1)
22	25549	449	518(494,555)	510(485,554)	0.996 595	4.52(4.50,4.54)	0.022 9(0.022 3,0.023 6)
28	25549	293	370(341,416)	371(335,435)	0.997 221	3.24(3.22,3.26)	0.103 5(0.101 1,0.105 8)
33	25549	76	239(187,314)	176(119,309)	0.998 317	0.43(0.41,0.44)	0.803 3(0.797 1,0.809 4)
34	25549	28	193(129,297)	79(43,202)	0.999 295	0.34(0.33,0.35)	0.823 6(0.817 7,0.829 5)
35	25549	57	155(115,222)	98(74,158)	0.998 748	0.41(0.40,0.42)	0.805 3(0.799 2,0.811 4)
36	25549	26	64(44,108)	46(31,99)	0.999 491	0.61(0.60,0.62)	0.643 0(0.637 1,0.648 9)
49	25549	165	193(178,226)	199(178,253)	0.998 904	3.57(3.55,3.59)	0.055 3(0.054 0,0.056 6)
50	25549	186	229(208,271)	234(207,296)	0.998 552	3.69(3.67,3.70)	0.044 1(0.043 3,0.045 0)
51	25549	206	242(225,276)	288(241,402)	0.998 474	3.71(3.69,3.72)	0.054 6(0.053 1,0.056 1)
52	25549	183	245(219,293)	238(210,294)	0.997 965	1.69(1.67,1.72)	0.466 0(0.458 7,0.473 3)
58	25549	178	216(198,251)	209(192,250)	0.998 630	2.94(2.91,2.96)	0.146 9(0.143 1,0.150 6)
59	25549	258	287(274,314)	290(273,329)	0.998 474	3.79(3.77,3.81)	0.047 0(0.045 9,0.048 0)
60	25549	272	318(299,351)	309(290,346)	0.997 965	3.93(3.92,3.95)	0.035 5(0.034 8,0.036 2)
61	25549	235	296(271,339)	324(280,409)	0.997 652	3.49(3.47,3.51)	0.060 5(0.059 1,0.061 9)

样本号	序列	Alpha 多样性
样本号	序列	OTU	Ace指数	Chao1指数	Coverage指数	Shannon指数	Simpson指数
23	25549	248	326(296,373)	317(286,374)	0.997 221	2.40(2.37,2.42)	0.244 1(0.239 4,0.248 7)
24	25549	313	366(345,401)	353(334,390)	0.997 652	4.17(4.15,4.18)	0.028 4(0.027 8,0.029 0)
25	25549	408	516(480,570)	523(477,601)	0.996 086	4.08(4.06,4.11)	0.042 8(0.041 6,0.044 0)
26	25549	435	492(472,524)	482(462,519)	0.996 986	4.25(4.23,4.27)	0.031 3(0.030 6,0.032 0)
27	25549	446	516(492,553)	512(485,559)	0.996 516	3.85(3.83,3.88)	0.066 3(0.064 3,0.068 2)
30	25549	439	508(484,545)	535(494,605)	0.996 595	4.42(4.40,4.44)	0.030 5(0.029 5,0.031 5)
31	25549	532	600(577,635)	600(572,646)	0.996 399	4.60(4.58,4.62)	0.026 9(0.026 1,0.027 6)
32	25549	264	331(305,376)	320(294,369)	0.997 534	3.36(3.34,3.38)	0.102 8(0.100 0,0.105 5)
39	25549	28	62(39,138)	88(45,244)	0.999 374	0.76(0.74,0.77)	0.605 3(0.599 0,0.611 6)
40	25549	21	40(26,94)	32(24,69)	0.999 609	0.94(0.93,0.95)	0.474 8(0.470 3,0.479 2)
65	25549	339	389(369,422)	404(373,462)	0.997 534	4.19(4.17,4.21)	0.035 0(0.033 9,0.036 0)