Abstract

• taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution.
  • High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions.

Background

• Two general strategies are commonly used to classify 16S rDNA reads

  1. taxonomic assignment approaches that utilize comprehensive databases (e.g., the Ribosomal Database Project (RDP), APB-Silva, and Greengenes)
  2. taxonomy-independent approaches that classify reads into Operational Taxonomic Units (OTUs)
  • problematic for taxonomic identification, in part, because these databases are incomplete and contain many unidentified and poorly annotated sequences.

• Here, we describe:

  1. the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for vaginal taxa; and
  2. STIRRUPS, a general method for species-level classification that involves three steps: database curation, clustering reference database sequences into species-level taxa, and taxonomic classification using the STIRRUPS Classifier.

  The method is validated on reads from replicates of a mock sample containing DNA from six vaginally relevant bacteria and applied to ~30 million V1-V3 16S rDNA reads from samples obtained from mid-vaginal swabs.

Method

• STIRRUPS : Species-level Taxon Identification of rDNA Reads using a USEARCH Pipeline Strategy

• STIRRUPS method is comprised

  1. curation of an appropriate database
  2. clustering the reference database sequences into species-level taxa
  3. axonomic classification using the STIRRUPS Classifier, a tool that employs the USEARCH algorithm

The Vaginal 16S rDNA Reference Database

• HMP - vaginal 사용
• V1-V3 영역 시퀀스에 MUSCLE로 msa 후 수동 검사
• BLAST검색을 수행
• For 87 of the targeted genera, we sought at least one representative 16S rDNA sequence for every validly named bacterial species in the genus from the sequences available in GenBank.
  • 30 %기록 후 제외
    • 기준: Note: many of these excluded species are not commonly found in vaginal samples; and therefore, the exclusion of these organisms does not greatly impact the use of the database to characterize reads from vaginal samples.
  • Selected representative sequences contain no more than five ambiguous bases in the V1-V3 region and span the entire V1-V3 window, which has an average length of ~485 bases.
  • Nucleotide-nucleotide BLAST searches against the non-redundant GenBank nucleotide database were performed to ensure selection of representative reference sequences, and UCHIME de novo chimera detection was used to identify and remove chimeric sequences.

Sequence variation plot

• CLUSTALW로 16S rDNA의 V1-V3 영역의 msa → 보조자료 123 확인
• each nucleotide (ACTG) at every position was calculated using Mesquite
  • gap characters were ignored
• shnnon's diversity index was calculated as :
  • $H(x) = -\sum_{i=A,C,T,G} p(x_i)lnp(x_i)$ , where $p(x_i)$ represents the probability of each base i at position x