三种DNA提取方法对半滑舌鳎肠道菌群高通量测序结果的影响

doi:10.19640/j.cnki.jtau.2022.03.006

Journal of Tianjin Agricultural University ›› 2022, Vol. 29 ›› Issue (3): 29-35.doi: 10.19640/j.cnki.jtau.2022.03.006

• Researches and Scientific Notes • Previous Articles Next Articles

Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing

Ge Lunhua, Jiang Boyun, Sun Jingfeng^{Corresponding Author}, Lü Aijun, Hu Xiucai, Chen Limei

College of Fisheries, Tianjin Agricultural University, Tianjin 300392, China

Received:2020-07-24 Online:2022-09-30 Published:2022-10-10

Abstract

Abstract: The purpose of this study was to analyze the effects of different DNA extraction methods on structural analysis of intestinal microbiota from C. semilaevis based on high throughput sequencing and comprehensively learn about the structural characteristics of its intestinal flora. In this study, three DNA extraction methods including combination of lysozyme and ultrasonic lysis（CLU）, Zirmil-beating cell disruption（ZBC）and QIA commercial kits were used to extract the genomic DNA of intestinal microflora from healthy C. semilaevis. The DNA samples were sequenced on an Illumina MiSeq high-throughput sequencing platform, and the structural characteristics and diversities of the intestinal microflora were analyzed. The results showed that a total of 7 516 OTUs were obtained across all samples extracted by the three DNA extraction methods. The identified dominant bacterial taxa were similar among these samples. At Phylum level, the dominant bacterial taxa included Proteobacteria, Firmicutes, Spirochaetae and Bacteroides. Through Alpha diversity index analysis, the corresponding Chao1 indexes of CLU, ZBC and QIA methods were 5 668, 5 868 and 4 090 respectively, and the Simpson index were 0.021, 0.022 and 0.018 respectively. The Chao1 and Simpson indexes of QIA methods were all lower than those of other methods. Beta diversity analysis based on the UniFrac distance matrix showed that the microbial community structure and species richness were more similar between the CLU and QIA samples. These results indicated that these three different DNA extraction methods reached the similar results in the major dominant intestinal microflora, based on the investigation using high-throughput sequencing analysis. However, biases occurred in the identification results of some less abundant intestinal microflora. The results providereference for research methods of the diversity and structure of fish intestinal microflora, especially the DNA extraction strategy, and contribute to comprehensively expounding the structural characteristics of intestinal microflora of C. semilaevis.

Key words: high-throughput sequencing, DNA extraction method, C. semilaevis, intestinal microflora, composition structure

CLC Number:

S917.1

Ge Lunhua, Jiang Boyun, Sun Jingfeng, Lü Aijun, Hu Xiucai, Chen Limei. Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing[J]. Journal of Tianjin Agricultural University, 2022, 29(3): 29-35.

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: http://xuebao.tjau.edu.cn/EN/10.19640/j.cnki.jtau.2022.03.006

http://xuebao.tjau.edu.cn/EN/Y2022/V29/I3/29

References

[1] 邓思平,陈松林,田永胜,等. 半滑舌鳎的性腺分化和温度对性别决定的影响[J]. 中国水产科学,2007,14(5):714-719.
[2] 陈君. 工厂化循环水养殖半滑舌鳎主要细菌性疾病及其控制[D]. 上海:上海海洋大学,2012.
[3] CHAIYAPECHARA S,RUNGRASSAMEE W,SURIYACHAY I,et al.Bacterial community associated with the intestinal tract of P. monodon in commercial farms[J]. Microbial Ecology,2012,63(4):938-953.
[4] SCHRYVER P,VADSTEIN O.Ecological theory as a foundation to control pathogenic invasion in aquaculture[J]. The ISME Journal:Multidisciplinary Journal of Microbial Ecology,2014,8(12):2360-2368.
[5] 张正,廖梅杰,李彬,等. 两种疾病发生对养殖半滑舌鳎肠道菌群结构的影响分析[J]. 水产学报,2014,38(9):1565-1572.
[6] 王一泽. 饲料中添加抗生素对半滑舌鳎肠道酶组织化学、肠道转录组及菌群结构的影响[D]. 天津:天津农学院,2017.
[7] 汪林庆. 半滑舌鳎肠道微生物及C9基因在免疫反应中的应答特征[D]. 福州:福州大学,2017.
[8] XIA J H,LIN G,FU G H,et al.The intestinal microbiome of fish under starvation[J]. BMC Genomics,2014,15(1):266-277.
[9] NADARAJAN K M,HO G G,JAEWON R,et al.Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil[J]. Journal of Microbiology,2015,53(11):767-775.
[10] 郑艺,张家超,郭壮,等. 基于高通量测序技术分析肠道菌群及其影响因素的研究进展[J]. 中国食品学报,2014,14(11):157-164.
[11] 秦楠,栗东芳,杨瑞馥. 高通量测序技术及其在微生物学研究中的应用[J]. 微生物学报,2011,51(4):445-457.
[12] 王晓璐,李乐,王友红,等. 南非斑节对虾肠道组织结构及菌群特征、代谢活性研究[J]. 广西科学院学报,2019,35(4):325-331.
[13] 刘志刚,卢迈新,可小丽,等. 尼罗罗非鱼肠道及养殖环境中菌群结构与链球菌病的相关性[J]. 水产学报,2018,42(10):1635-1647.
[14] 郭政宏,舒邦杰,严亨秀. 藏山羊粪便总DNA不同提取方法的比较[J]. 黑龙江畜牧兽医,2016,26(4):93-96.
[15] ABRIOUEL H,BEN O N,LOPEZ R L,et al.Culture- independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods[J]. International Journal of Food Microbiology,2006,111(3):228-233.
[16] 万晶晶,张汝嘉,邢德峰,等. 超声波破碎法提取活性污泥DNA及其DGGE分析[J]. 哈尔滨工业大学学报,2008,40(4):559-562.
[17] ZOETENDAL E G,HEILIG H G,KLAASSENS E S,et al.Isolation of DNA from bacterial samples of the human gastrointestinal tract[J]. Nature Protocols,2006,1(2):870-873.
[18] 许朋,郑春辉,孙智勇,等. 磁珠法快速提取基因组DNA的实验研究[J]. 生物信息学,2018,16(3):190-195.
[19] RUBIN B E R,SANDERS J G,HAMPTON-MARCELL J,et al. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure[J]. MicrobiologyOpen,2014,3(6):910-921.
[20] 温崇庆,何瑶瑶,薛明,等. 高通量测序分析DNA提取引起的对虾肠道菌群结构偏差[J]. 微生物学报,2016,56(1):130-142.
[21] HAN Z,SUN J,LU A,et al.Biases from different DNA extraction methods in intestine microbiome research based on 16S rDNA sequencing:a case in the koi carp,Cyprinus carpio var. Koi[J]. MicrobiologyOpen,2019,8(1):e626.
[22] HAN Z,SUN J,LU A,et al.A modified method for genomic DNA extraction from the fish intestinal microflora[J]. AMB Express,2018,8(1):52-60.
[23] JIANG B,SUN J,LÜ A,et al.Impact of DNA extraction methods on the observed microbial communities from the intestinal flora of the penaeid shrimp Litopenaeus vannamei[J]. FEMS Microbiology Letters,2019,366(9):99.
[24] TANJA M,SALZBERG S L.FLASH:fast length adjustment of short reads to improve genome assemblies[J]. Bioinformatics,2011,27(21):2957-2963.
[25] EDGAR R C,HAAS B J,CLEMENTE J C,et al.UCHIME improves sensitivity and speed of chimera detection[J]. Bioinformatics,2011,27(16):2194-2200.
[26] WANG Q,GARRITY G M,TIEDJE J M,et al.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied and Environmental Microbiology,2007,73(16):5261-5267.
[27] ZHOU J,WU L,DENG Y,et al.Reproducibility and quantitation of amplicon sequencing-based detection[J]. The ISME Journal:Multidisciplinary Journal of Microbial Ecology,2011,5(8):1303-1313.
[28] 苏双良,王慧,李洪萍,等. 两种不同土鳖虫肠道菌群总DNA提取方法的比较及ERIC-PCR指纹图谱分析[J]. 动物医学进展,2014,35(5):85-89.
[29] FRIEDRICH V W,GÖBEL U B,ERKO S. Determination of microbial diversity in environmental samples:pitfalls of PCR-based rRNA analysis[J]. FEMS Microbiology Reviews,1997,3:213-229.
[30] 郑璐,高乃云. 环境样品中DNA提取方法的研究进展[J]. 给水排水,2010,46(5):175-178.
[31] CHEN H,RANGASAMY M,TAN S Y,et al.Evaluation of five methods for total DNA extraction from western corn rootworm beetles[J]. PLoS One,2010,5(8):e11963.
[32] 宋娟. 溶菌酶的分离方法及进展[J]. 河南化工,2017,34(7):16-18.
[33] YU L P,SUN B G,LI J,et al.Characterization of a c-type lysozyme of Scophthalmus maximus:expression,activity,and antibacterial effect[J]. Fish & Shellfish Immunology,2013,34(1):46-54.
[34] 张正,李彬,王印庚,等. 基于高通量测序的池塘养殖半滑舌鳎消化道菌群的结构特征分析[J]. 水生生物学报,2015,39(1):38-45.
[35] WU S,TIAN J,GATESOUPE F.et al.Intestinal microbiota of gibel carp(Carassius auratus gibelio)and its origin as revealed by 454 pyrosequencing[J]. World Journal of Microbiology & Biotechnology,2013,29(9):1585-1595.
[36] 张燕玉,韩卓然,孙敬锋,等. 海藻希瓦氏菌感染对半滑舌鳎肠道菌群结构及相关功能基因表达的影响[J]. 南方农业学报,2019,50(10):2300-2307.

Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing

PDF

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 8

Recommended Articles

Metrics

Comments

[1]	Chen Xuan, Zhang Dajuan, Zhang Shulin, Wang Zebin, Dai Wei, Bi Xiangdong. The competition and allelopathy of Microcystis aeruginosa with Cyanoidetes longissima and Scenedesmus quadricauda [J]. Journal of Tianjin Agricultural University, 2023, 30(4): 32-38.
[2]	Wang Xiaoyu, Ren Shutian, Wu Yating, Wu Zun, Gao Haofeng, Shao Peng. Isolation of symbiotic bacteria of Euglena gracilis and effects on the growth of algal cells [J]. Journal of Tianjin Agricultural University, 2022, 29(2): 57-62.
[3]	Gu Xueqin, Wang Qingkui, Wang Yang. Research progress on bacterial community structure in recirculating aquaculture system for marine fish [J]. Journal of Tianjin Agricultural University, 2022, 29(2): 76-84.
[4]	YE Ming-hao, HU Xiu-cai, LÜ Ai-jun, SUN Jing-feng, LIU Xiao-xue, BAI Jie. Isolation, identification and antibiotic sensitivity of the pathogenic Shewanella putrefaciens from the grass carp Ctenopharyngodon idellus [J]. Journal of Tianjin Agricultural University, 2018, 25(4): 52-57.
[5]	ZHAN Hai, WANG Guang-jun, CHEN Cheng-xun. Identification and Denitrification Ability of an Aerobic Denitrifier [J]. Journal of Tianjin Agricultural University, 2017, 24(2): 44-48.
[6]	HU Xuan, SUN Jing-feng, CHEN Cheng-xun, XING Ke-zhi. Isolation and Identification of Pathogen of Ascites Disease from Cynoglossus semilaevis Günther and Its Antibiotic Sensitivity Analysis [J]. Journal of Tianjin Agricultural University, 2014, 21(3): 12-16.
[7]	SUN Cheng-bo, MIAO Jian-sheng, HAN Zhi-hui, ZHAO Dong-yan, LI Jian-guo, RONG Hai-yan. Phytoplankton Analysis Based on Multiple Bio-treatment System [J]. Journal of Tianjin Agricultural University, 2012, 19(3): 52-57.
[8]	PENG Hui-hui, ZHANG Kai, CHEN Cheng-xun, XIE Jun, LI Zhi-fei, YU Er-meng, LIU Long-fei, GONG Wang-bao, TIAN Jing-jing, WANG Guang-jun. Effects of carp（Cyprinus carpio）on phytoplankton and environmental factors in paddy fields [J]. Journal of Tianjin Agricultural University, 2020, 27(1): 37-43.