PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample

被引：442

作者：

Acinas, SG ^{[1
]}

Sarma-Rupavtarm, R ^{[1
]}

Klepac-Ceraj, V ^{[1
]}

Polz, MF ^{[1
]}

机构：

[1] MIT, Dept Civil & Environm Engn, Cambridge, MA 02139 USA

来源：

APPLIED AND ENVIRONMENTAL MICROBIOLOGY | 2005年 / 71卷 / 12期

关键词：

D O I：

10.1128/AEM.71.12.8966-8969.2005

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

引用

页码：8966 / 8969

页数：4

共 38 条

[1] Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons [J].

Acinas, SG ;

Marcelino, LA ;

Klepac-Ceraj, V ;

Polz, MF .

JOURNAL OF BACTERIOLOGY, 2004, 186 (09) :2629-2635

[2] Fine-scale phylogenetic architecture of a complex bacterial community [J].

Acinas, SG ;

Klepac-Ceraj, V ;

Hunt, DE ;

Pharino, C ;

Ceraj, I ;

Distel, DL ;

Polz, MF .

NATURE, 2004, 430 (6999) :551-554

[3] CHIMERIC CDNA CLONES - A NOVEL PCR ARTIFACT [J].

BRAKENHOFF, RH ;

SCHOENMAKERS, JGG ;

LUBSEN, NH .

NUCLEIC ACIDS RESEARCH, 1991, 19 (08) :1949-1949

[4] PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases [J].

Cline, J ;

Braman, JC ;

Hogrefe, HH .

NUCLEIC ACIDS RESEARCH, 1996, 24 (18) :3546-3551

[5] The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis [J].

Cole, JR ;

Chai, B ;

Farris, RJ ;

Wang, Q ;

Kulam, SA ;

McGarrell, DM ;

Garrity, GM ;

Tiedje, JM .

NUCLEIC ACIDS RESEARCH, 2005, 33 :D294-D296

[6] Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization [J].

Cottrell, MT ;

Kirchman, DL .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2000, 66 (12) :5116-5122

[7]

Fisher MM, 1999, APPL ENVIRON MICROB, V65, P4630

[8] Bacterial community structure associated with a dimethylsulfoniopropionate-producing North Atlantic algal bloom [J].

González, JM ;

Simó, R ;

Massana, R ;

Covert, JS ;

Casamayor, EO ;

Pedrós-Alió, C ;

Moran, MA .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2000, 66 (10) :4237-4246

[9] THE POPULATION FREQUENCIES OF SPECIES AND THE ESTIMATION OF POPULATION PARAMETERS [J].

GOOD, IJ .

BIOMETRIKA, 1953, 40 (3-4) :237-264

[10] Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases [J].

Hugenholtz, P ;

Huber, T .

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2003, 53 :289-293

← 1 2 3 4 →