Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.
The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.
Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada. heather.maughan@utoronto.ca
Heather
H
Maughan
Pauline W
PW
Wang
Julio
J
Diaz Caballero
Pauline
P
Fung
Yunchen
Y
Gong
Sylva L
SL
Donaldson
Lijie
L
Yuan
Shaf
S
Keshavjee
Yu
Y
Zhang
Yvonne C W
YC
Yau
Valerie J
VJ
Waters
D Elizabeth
DE
Tullis
David M
DM
Hwang
David S
DS
Guttman
D001419:Bacteria / Q000145:classification / Q000235:genetics*; D003198:Computer Simulation; D003550:Cystic Fibrosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008168:Lung / Q000382:microbiology / Q000473:pathology; D054892:Metagenome / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013183:Sputum / Q000382:microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S