Effect of Different Pretreatment on the Results of High-throughput Sequencing of Microbial Community in Mouse Feces

doi:10.19586/j.2095-2341.2022.0158

Current Biotechnology ›› 2023, Vol. 13 ›› Issue (1): 115-123.DOI: 10.19586/j.2095-2341.2022.0158

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Effect of Different Pretreatment on the Results of High-throughput Sequencing of Microbial Community in Mouse Feces

Xin LIU¹(), Huiling HU¹, Minai ZHANG², Guozhen XUE¹, Xiaorui YAN¹, Xichun ZHANG¹(), Jiquan LIU¹()

^1.School of Traditional Chinese Medicine and Food Engineering，Shanxi University of Traditional Chinese Medicine，Shanxi Jinzhong 030600，China
^2.Customs Technical Center of Taiyuan，Taiyuan 030021，China

Received:2020-09-08 Accepted:2020-11-30 Online:2023-01-25 Published:2023-02-07
Contact: Xichun ZHANG,Jiquan LIU

不同预处理对小鼠粪样菌群高通量测序分析结果的影响

刘昕¹(), 胡会玲¹, 张敏爱², 薛帼珍¹, 闫晓睿¹, 张希春¹(), 刘计权¹()

^1.山西中医药大学中药与食品工程学院，山西晋中 030600
^2.太原海关技术中心，太原 030021

通讯作者: 张希春,刘计权
作者简介:刘昕 E-mail:1113984315@qq.com；
基金资助:
山西省自然科学基金面上项目(20210302123233);山西省现代农业产业技术体系建设专项资金(2021-11);山西中医药大学科技创新科学团队项目(2018TD-009);山西中医药大学科技创新培育计划项目(2022PY-TH-30)

Abstract

Abstract:

High-throughput sequencing was used to compare the effect of different pretreatments on faecal flora structure in mice， so as to provide reference for further research. Fresh fecal samples of Kunming mice were collected and divided into three groups： original fecal sample group， saline treatment group and PBS treatment group. Fecal DNA was extracted， sequenced using the Illumina MiSeq platform， and 16S rRNA V3~V4 region gene libraries of three sets of samples were analyzed by bioinformatics analysis. The results showed that there were 188 common OTUs in the three groups， and different pretreatments had an effect on faecal microbial diversity and abundance. In saline treatment group， PBS treatment group and original fecal sample group， 14， 11 and 12 phyla were detected and there were 11 common phyla in all three groups； 24， 21 and 21 classes were detected and there were 20 common classes in all three groups； 62， 55 and 59 families were detected， there were 26 common families in all three groups； 147， 126 and 137 genus were detected， there were 117 common genus in all three groups. Overall， the saline treatment group had the highest relative microbial diversity and abundance， followed by the original fecal sample group， and the PBS treatment group had the lowest. Saline treatment can improve the accuracy of intestinal flora detection to a certain extent.

Key words: high-throughput sequencing, saline solution, PBS buffer, fecal microbial communityretreatment

摘要：

应用高通量测序技术比较不同预处理对小鼠粪样菌群结构的影响，以期为后续相关研究提供参考依据。采集昆明小鼠新鲜粪样，分为原始粪样组、生理盐水处理组和PBS处理组，提取粪样DNA，采用Illumina MiSeq平台进行测序对3组样本的16S rRNA V3~V4区基因文库进行生物信息学分析。结果显示，3组样本拥有共同的OTUs 188个，不同预处理对粪样微生物多样性和丰度产生影响，生理盐水处理组、PBS处理组和原始粪样组分别检出14、11、12个门，3组样本共有门11个；分别检出24、21、21个纲，3组样本共有纲20个；分别检出62、55、59个科，3组样本共有科26个；分别检出147、126、137个属，3组样本共有属117个。总体来看，生理盐水处理组微生物多样性和丰度相对最高，原始粪样组次之，PBS处理组最低。粪样经生理盐水处理后，能在一定程度上提高肠道菌群检测的准确性。

关键词: 高通量测序, 生理盐水, PBS缓冲液, 粪样菌群

CLC Number:

R965

Xin LIU, Huiling HU, Minai ZHANG, Guozhen XUE, Xiaorui YAN, Xichun ZHANG, Jiquan LIU. Effect of Different Pretreatment on the Results of High-throughput Sequencing of Microbial Community in Mouse Feces[J]. Current Biotechnology, 2023, 13(1): 115-123.

刘昕, 胡会玲, 张敏爱, 薛帼珍, 闫晓睿, 张希春, 刘计权. 不同预处理对小鼠粪样菌群高通量测序分析结果的影响[J]. 生物技术进展, 2023, 13(1): 115-123.

Figures/Tables 15

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样本编号	Clean-tags	Valid-tags	Valid-tags mean length/bp	OTUs
S1	76 432	63 910	412.69	656
S2	77 145	65 163	412.34	661
S3	77 400	65 576	412.63	670
P1	72 165	61 166	419.79	351
P2	75 419	65 217	420.02	373
P3	75 107	65 796	421.21	368
Y1	75 933	64 761	419.98	442
Y2	69 909	56 015	420.04	424
Y3	77 752	65 465	419.02	436

样本编号	Clean-tags	Valid-tags	Valid-tags mean length/bp	OTUs
S1	76 432	63 910	412.69	656
S2	77 145	65 163	412.34	661
S3	77 400	65 576	412.63	670
P1	72 165	61 166	419.79	351
P2	75 419	65 217	420.02	373
P3	75 107	65 796	421.21	368
Y1	75 933	64 761	419.98	442
Y2	69 909	56 015	420.04	424
Y3	77 752	65 465	419.02	436

组别	Chao 1	Observed-species	Simpson	Shannon	Goods coverage
S组	707.32 ± 14.090a	650.17 ± 5.500a	0.94 ± 0.001a	5.59 ± 0.030a	0.998 379a
P组	418.67 ± 10.430c	353.07 ± 8.110c	0.78 ± 0.010c	3.42 ± 0.050c	0.998 581a
Y组	496.76 ± 12.680b	424.83 ± 3.820b	0.85 ± 0.020b	4.12 ± 0.050b	0.998 225a

组别	Chao 1	Observed-species	Simpson	Shannon	Goods coverage
S组	707.32 ± 14.090a	650.17 ± 5.500a	0.94 ± 0.001a	5.59 ± 0.030a	0.998 379a
P组	418.67 ± 10.430c	353.07 ± 8.110c	0.78 ± 0.010c	3.42 ± 0.050c	0.998 581a
Y组	496.76 ± 12.680b	424.83 ± 3.820b	0.85 ± 0.020b	4.12 ± 0.050b	0.998 225a

组别	S组	P组	Y组
拟杆菌门	0.469 ± 0.003a	0.455 ± 0.013a	0.415 ± 0.056a
厚壁菌门	0.478 ± 0.007a	0.151 ± 0.031c	0.251 ± 0.035b
变形菌门	0.049 ± 0.005b	0.388 ± 0.046a	0.330 ± 0.025a
放线菌门	0.001 ± 0.000b	0.004 ± 0.002a	0.003 ± 0.000ab
脱铁杆菌门	0.001 ± 0.000a	0.001± 0.000a	0.001 ± 0.000a

Effect of Different Pretreatment on the Results of High-throughput Sequencing of Microbial Community in Mouse Feces

不同预处理对小鼠粪样菌群高通量测序分析结果的影响

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Figures/Tables 15

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组别	S组	P组	Y组
拟杆菌纲	0.469 ± 0.003a	0.455 ± 0.013a	0.415 ± 0.056a
梭菌纲	0.464 ± 0.007a	0.133 ± 0.027b	0.169 ± 0.016b
γ-变形菌纲	0.046 ± 0.005a	0.387 ± 0.046b	0.329 ± 0.025b
丹毒丝菌纲	0.001 ± 0.001b	0.006 ± 0.001b	0.074 ± 0.056a
厚壁菌纲	0.012 ± 0.000a	0.007 ± 0.002b	0.002 ± 0.001c

组别	S组	P组	Y组
普雷沃氏菌科	0.397 ± 0.004a	0.446 ± 0.014a	0.373 ± 0.061a
肠杆菌科	0.010 ± 0.001b	0.322 ± 0.026a	0.314 ± 0.038a
毛螺菌科	0.210 ± 0.006a	0.104 ± 0.026b	0.125 ± 0.02b
瘤胃菌科	0.240 ± 0.010a	0.028 ± 0.002b	0.042 ± 0.004c
伯克霍尔德科	0.034 ± 0.004a	0.065 ± 0.019b	0.014 ± 0.014a
拟杆菌科	0.046 ± 0.001a	0.007 ± 0.001c	0.037 ± 0.008b
丹毒杆菌科	0.010 ± 0.000a	0.006 ± 0.001b	0.074 ± 0.055a

组别	S组	P组	Y组
普雷沃氏菌属9	0.387 ± 0.004a	0.445 ± 0.014a	0.372 ± 0.061a
克雷伯氏杆菌属	0.007 ± 0.000b	0.133 ± 0.045a	0.188 ± 0.038a
大肠杆菌-志贺氏菌属	0.003 ± 0.001c	0.176 ± 0.019a	0.113 ± 0.008b
罗氏菌属	0.120 ± 0.007a	0.006 ± 0.001b	0.024 ± 0.018b
粪杆菌属	0.089 ± 0.006a	0.013 ± 0.003b	0.010 ± 0.004b
副萨特氏菌属	0.033 ± 0.005b	0.065 ± 0.019a	0.014 ± 0.014b
拟杆菌属	0.044 ± 0.002a	0.007 ± 0.001b	0.037 ± 0.008a
链状杆菌属	0.000 ± 0.000b	0.005 ± 0.001b	0.074 ± 0.056a
毛螺旋菌科UCG-004菌属	0.000±0.000b	0.027 ± 0.008a	0.023 ± 0.004a
乳梭菌属	0.003 ± 0.000b	0.022 ± 0.003a	0.022 ± 0.003a

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