Abstract
High-throughput sequencing of 16S rRNA gene amplicons has revolutionized the capacity and depth of microbial community profiling. Several sequencing platforms are available, but most phylogenetic studies are performed on the 454-pyrosequencing platform because its longer reads can give finer phylogenetic resolution. The Pacific Biosciences (PacBio) sequencing platform is significantly less expensive per run, does not rely on amplification for library generation, and generates reads that are, on average, four times longer than those from 454 (C2 chemistry), but the resulting high error rates appear to preclude its use in phylogenetic profiling. Recently, however, the PacBio platform was used to characterize four electrosynthetic microbiomes to the genus-level for less than USD 1,000 through the use of PacBio’s circular consensus sequence technology. Here, we describe in greater detail: 1) the output from successful 16S rRNA gene amplicon profiling with PacBio, 2) how the analysis was contingent upon several alterations to standard bioinformatic quality control workflows, and 3) the advantages and disadvantages of using the PacBio platform for community profiling.
Highlights
Strand orientation Unlike sequences originating from 454, the user cannot control which template strand (+/−) is sequenced in Pacific Biosciences (PacBio)
The phylogenetic profiling of microbial communities using 16S ribosomal RNA (rRNA) gene amplicon sequencing is a routine practice in microbial ecology
The size of the 16S rRNA gene V1-V3 or V2-V3 amplicons gave sufficient single-molecule coverage to produce full-amplicon-length ccs reads with an average Phred quality score of 60 (1 in 1,000,000 probability of an incorrect call at each base) after quality control (QC) (Figure 2)
Summary
The phylogenetic profiling of microbial communities using 16S rRNA gene amplicon sequencing is a routine practice in microbial ecology. Since phylogenetic profiling requires high read accuracy, low quality reads are problematic, but this issue can be alleviated through the use of PacBio circular consensus sequencing (ccs). The size of the 16S rRNA gene V1-V3 (approximately 515 bp, bacteria) or V2-V3 (approximately 400 bp, archaea) amplicons gave sufficient single-molecule coverage (using C2 chemistry and a 45 min movie length) to produce full-amplicon-length ccs reads with an average Phred quality score of 60 (1 in 1,000,000 probability of an incorrect call at each base) after quality control (QC) (Figure 2). Barcode sequence, fewer than a specified number of bases, or chimeras [5] As this workflow was not wholly sufficient for use with PacBio-generated data output [4], the necessary alterations are discussed further in the text, all of which can be executed with open-source software such as mothur [6] or QIIME [7]. 100 bp for primer (archaea) barcode 1 for primer (bacteria) barcode 5 rev primer (universal) archaeal amplicon bacterial amplicon
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