The cervical microbiome over 7 years and a comparison of methodologies for its characterization.
BACKGROUND
The rapidly expanding field of microbiome studies offers investigators a large choice of methods for each step in the process of determining the microorganisms in a sample. The human cervicovaginal microbiome affects female reproductive health, susceptibility to and natural history of many sexually transmitted infections, including human papillomavirus (HPV). At present, long-term behavior of the cervical microbiome in early sexual life is poorly understood.


METHODS
The V6 and V6-V9 regions of the 16S ribosomal RNA gene were amplified from DNA isolated from exfoliated cervical cells. Specimens from 10 women participating in the Natural History Study of HPV in Guanacaste, Costa Rica were sampled successively over a period of 5-7 years. We sequenced amplicons using 3 different platforms (Sanger, Roche 454, and Illumina HiSeq 2000) and analyzed sequences using pipelines based on 3 different classification algorithms (usearch, RDP Classifier, and pplacer).


RESULTS
Usearch and pplacer provided consistent microbiome classifications for all sequencing methods, whereas RDP Classifier deviated significantly when characterizing Illumina reads. Comparing across sequencing platforms indicated 7%-41% of the reads were reclassified, while comparing across software pipelines reclassified up to 32% of the reads. Variability in classification was shown not to be due to a difference in read lengths. Six cervical microbiome community types were observed and are characterized by a predominance of either G. vaginalis or Lactobacillus spp. Over the 5-7 year period, subjects displayed fluctuation between community types. A PERMANOVA analysis on pairwise Kantorovich-Rubinstein distances between the microbiota of all samples yielded an F-test ratio of 2.86 (p<0.01), indicating a significant difference comparing within and between subjects' microbiota.


CONCLUSIONS
Amplification and sequencing methods affected the characterization of the microbiome more than classification algorithms. Pplacer and usearch performed consistently with all sequencing methods. The analyses identified 6 community types consistent with those previously reported. The long-term behavior of the cervical microbiome indicated that fluctuations were subject dependent.
Department of Pediatrics, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, United States of America.
Benjamin C
BC
Smith
Thomas
T
McAndrew
Zigui
Z
Chen
Ariana
A
Harari
David M
DM
Barris
Shankar
S
Viswanathan
Ana Cecilia
AC
Rodriguez
Phillip
P
Castle
Rolando
R
Herrero
Mark
M
Schiffman
Robert D
RD
Burk
D002584:Cervix Uteri / Q000382:microbiology*; D016000:Cluster Analysis; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054892:Metagenome; D058889:Molecular Typing / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S