PacBio sequencing of 16S-ITS-23S rRNA operon amplicons

  • Joran Martijn (Creator)
  • Anders E. Lind (Creator)
  • Max E. Schön (Creator)
  • Ian Spiertz (Creator)
  • Lina Juzokaite (Creator)
  • Ignas Bunikis (Creator)
  • Olga V. Pettersson (Creator)
  • Thijs Ettema (Creator)



Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information for confident deep phylogenetic placements. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286 - 37850 raw ccs reads). Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with sequences similar to 250 bp amplicons of the 16S rRNA gene and identified several novel prokaryotic lineages. Our method allows users to obtain good quality, near full-length 16S and 23S rRNA gene sequences from environmental taxa and determines their phylogenetic context more confidently compared to short-read 16S rRNA gene sequencing methods.
Date made available26 Oct 2018

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