Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary | Zendy

Clarisse Marotz | Zendy; Anukriti Sharma | Zendy; Greg Humphrey | Zendy; Neil Gottel | Zendy; Christopher Daum | Zendy; Jack A. Gilbert | Zendy; Emiley A. EloeFadrosh | Zendy; Rob Knight | Zendy

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Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary

Author(s) -

Clarisse Marotz,

Anukriti Sharma,

Greg Humphrey,

Neil Gottel,

Christopher Daum,

Jack A. Gilbert,

Emiley A. EloeFadrosh,

Rob Knight

Publication year - 2019

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/btn-2018-0192

Subject(s) - amplicon , amplicon sequencing , biology , replicate , polymerase chain reaction , 16s ribosomal rna , computational biology , gene , genetics , microbiology and biotechnology , statistics , mathematics

Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.

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