Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis

被引：214

作者：

Wu, Liyou ^{[1
,2
]}

Wen, Chongqing ^{[1
,2
,4
]}

Qin, Yujia ^{[1
,2
]}

Yin, Huaqun ^{[1
,2
,5
,6
]}

Tu, Qichao ^{[1
,2
]}

Van Nostrand, Joy D. ^{[1
,2
]}

Yuan, Tong ^{[1
,2
]}

Yuan, Menting ^{[1
,2
]}

Deng, Ye ^{[1
,2
,8
]}

Zhou, Jizhong ^{[1
,2
,3
,7
]}

机构：

[1] Univ Oklahoma, Inst Environm Genom, Norman, OK 73019 USA

[2] Univ Oklahoma, Dept Microbiol & Plant Biol, Norman, OK 73019 USA

[3] Tsinghua Univ, Sch Environm, State Key Joint Lab Environm Simulat & Pollut Con, Beijing 100084, Peoples R China

[4] Guangdong Ocean Univ, Coll Fisheries, Zhanjiang, Guangdong, Peoples R China

[5] Cent S Univ, Sch Minerals Proc & Bioengn, Changsha, Hunan, Peoples R China

[6] Minist Educ, Key Lab Biomet, Changsha, Hunan, Peoples R China

[7] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USA

[8] Chinese Acad Sci, Res Ctr Ecoenvironm Sci, CAS Key Lab Environm Biotechnol, Beijing, Peoples R China

来源：

BMC MICROBIOLOGY | 2015年 / 15卷

基金：

美国国家科学基金会;

关键词：

Next generation sequencing; Low diversity sample; Amplicon sequencing; Illumina Miseq; Microbial community; Phasing primer; Microbial ecology; CARBON-DIOXIDE; GENERATION; DIVERSITY; PATTERNS; DEPTH;

D O I：

10.1186/s12866-015-0450-4

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

Background: Although high-throughput sequencing, such as Illumina-based technologies (e.g. MiSeq), has revolutionized microbial ecology, adaptation of amplicon sequencing for environmental microbial community analysis is challenging due to the problem of low base diversity. Results: A new phasing amplicon sequencing approach (PAS) was developed by shifting sequencing phases among different community samples from both directions via adding various numbers of bases (0-7) as spacers to both forward and reverse primers. Our results first indicated that the PAS method substantially ameliorated the problem of unbalanced base composition. Second, the PAS method substantially improved the sequence read base quality (an average of 10 % higher of bases above Q30). Third, the PAS method effectively increased raw sequence throughput (similar to 15 % more raw reads). In addition, the PAS method significantly increased effective reads (9-47 %) and the effective read sequence length (16-96 more bases) after quality trim at Q30 with window 5. In addition, the PAS method reduced half of the sequencing errors (0.54-1.1 % less). Finally, two-step PCR amplification of the PAS method effectively ameliorated the amplification biases introduced by the long barcoded PCR primers. Conclusion: The developed strategy is robust for 16S rRNA gene amplicon sequencing. In addition, a similar strategy could also be used for sequencing other genes important to ecosystem functional processes

引用

页数：12