Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform

• Published in: Journal of microbiological methods 2012-10, Vol.91 (1), p.80-88
• Main Authors: Whiteley, Andrew S., Jenkins, Sasha, Waite, Ian, Kresoje, Nina, Payne, Hugh, Mullan, Bruce, Allcock, Richard, O'Donnell, Anthony
• Format: Article
• Language: English
• Subjects: anaerobic digesters; Archaea - classification; Archaea - genetics; Bacteria - classification; Bacteria - genetics; Bacteroides; Barcode multiplex; Biological and medical sciences; Biota; Clostridium; Community diversity; community structure; cost effectiveness; DNA Barcoding, Taxonomic - economics; DNA Barcoding, Taxonomic - methods; Fundamental and applied biological sciences. Psychology; genome; Ion Torrent; Metagenome; metagenomics; Metagenomics - economics; Metagenomics - methods; methanogens; microbial communities; Microbiology; PGM; pig manure; polymerase chain reaction; Polymerase Chain Reaction - economics; Polymerase Chain Reaction - methods; ribosomal RNA; RNA, Ribosomal, 16S - genetics; rRNA; sequence analysis; Sequence Analysis, DNA - economics; Sequence Analysis, DNA - methods; waste treatment; Waste Water - microbiology
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.07.008

Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun metagenomic approaches and generated 100,000+ to 1,000,000+ reads using ‘post-light’ based sequencing technology within different sized semi-conductor chips. Further development of Golay barcoded Ion Tags allowed multiplex analyses of microbial communities with substantially reduced costs compared with platforms such as 454/GS-FLX. Using these protocols we assessed the bacterial and archaeal dynamics within covered anaerobic digesters used to treat piggery wastes. Analysis of these sequence data showed that these novel methanogenic waste treatment systems are dominated by bacterial taxa, in particular Clostridium, Synergistia and Bacteroides that were maintained as a stable community over extended time periods. Archaeal community dynamics were more stochastic with the key methanogenic taxa more difficult to resolve, principally due to the poor congruence seen between community structures generated either by nested PCR or metagenomic approaches for archaeal analyses. Our results show that for microbial community structure and function analyses, the PGM platform provides a low cost, scalable and high throughput solution for both Tag sequencing and metagenomic analyses. ► ‘Post light’ DNA sequencer producing analyses in 8 hours with low reagent costs ► Scalable chip based sequencing ranging from 100,000 to 1,000,000+ reads ► Multiplex 16S rRNA analyses developed and demonstrated in anaerobic digesters. ► Novel covered anaerobic ponds dominated by Bacteroidia, Synergistia and Clostridia.