Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products

Abstract

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads allowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an observation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bacterial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.

Illumina-based analysis of microbial community diversity

“…Initial filtering removed 85% of the reads from run1 and 54% from run2 owing to errors stemming from several sources (Table 1), which is comparable to other studies that report filtering 40-70% of their initial reads (Gloor et al, 2010;Caporaso et al, 2011). We secured a total of 4.5 and 16.0 million paired-end reads for the two runs in spite of the high number of reads that were discarded because they did not meet our thresholds for accuracy; this, in turn yielded tens of thousands to millions of raw read-pairs per multiplexed sample (run1 x ¼ 380 253 ± 176 319; run2 x ¼ 3 936 966±2 299 439) ( Table 2).…”

“…Recent interest has focused on the applicability of other sequencing methodologies, most notably Solexa/Illumina (Lazarevic et al, 2009;Claesson et al, 2010;Gloor et al, 2010;Caporaso et al, 2011;Zhou et al, 2011), which is currently less than 1/100 the cost per read than 454 pyrosequencing. Although realizing much shorter read lengths, the Illumina technology can be tailored to yield sequences of increased lengths, as can be obtained by merging the paired-end reads generated from the same amplicon (Gloor et al, 2010;Rodrigue et al, 2010;Zhou et al, 2011). By integrating sample-identifying barcodes into the amplification primers, the Illumina platform, like 454 pyrosequencing, is amenable to a high level of multiplexing, which further increases its utility for examining large and complex sets of samples (Gloor et al, 2010).…”

“…Although realizing much shorter read lengths, the Illumina technology can be tailored to yield sequences of increased lengths, as can be obtained by merging the paired-end reads generated from the same amplicon (Gloor et al, 2010;Rodrigue et al, 2010;Zhou et al, 2011). By integrating sample-identifying barcodes into the amplification primers, the Illumina platform, like 454 pyrosequencing, is amenable to a high level of multiplexing, which further increases its utility for examining large and complex sets of samples (Gloor et al, 2010).…”

“…Individual samples were amplified, mixed and then used as templates to construct and sequence two standard Illumina paired-end libraries. Although Illumina sequence quality decays along the length of reads, our motivation for assembling paired-end reads was to increase the quality and confidence of the overlapping region (Gloor et al, 2010;Rodrigue et al, 2010;Zhou et al, 2011). Therefore the amplified V6 region was restricted to 100-160 bp to ensure an adequate overlap of the forward and reverse paired-end reads.…”

Microbiotes et métagénomique

“…Résultats des traitements (en rouge). [34][35][36]. Ces espèces qui paraissent minoritaires en nombre, peuvent contribuer à des processus spéci-fiques comme la méthanogenèse, la méthylotrophie, la fixation de l'azote dans les tubes digestifs et les rhizosphères [37][38][39] avec des répercussions majeures sur la nutrition, la croissance de l'hôte ou sur les cycles biogéochimiques [40].…”