© 2016Nature America, Inc. All rights reserved.
nature medicine 1
A major determinant of the microbiota composition of newborns
is the mode of delivery. Vaginally delivered infants harbor bacte-
rial communities resembling those of the maternal vagina, whereas
C-section–delivered infants are enriched in skin microbiota1.
The microbiome that colonizes the body of newborns can have a
determinant role in educating the immune system2. Early inter-
action with commensal microbes is essential for healthy immune
development and metabolic programming, and aberrant micro-
bial colonization in newborns has been associated with long-term
effects on host metabolism3 or impaired immune development2.
Epidemiological studies, although not showing causality, have
reported associations between C-section delivery and an increased
risk of obesity, asthma, allergies and immune deficiencies4–7. Rates
of cesarean delivery are increasing worldwide and in some countries
exceed 50% of total births8–10, a rate substantially greater than the
estimated 15% of births that require C-section delivery to protect
the health of the mother or baby11.
Here we exposed C-section–delivered infants to their maternal
vaginal fluids at birth and longitudinally determined the composition
of their microbiota to assess whether it developed more similarly to
vaginally born babies than to unexposed C-section–delivered infants.
We collected samples from 18 infants and their mothers, including 7
born vaginally and 11 delivered by scheduled C-section, of which four
were exposed to the maternal vaginal fluids at birth (Supplementary
Table 1). Briefly, the microbial restoration procedure, or vaginal micro-
bial transfer, consists of incubating sterile gauze in the vagina of moth-
ers who were negative for group B Streptococcus (GBS), had no signs
of vaginosis and had a vaginal pH < 4.5 during the hour preceding the
C-section. Within the first 2 min of birth, babies were exposed to their
maternal vaginal contents by being swabbed with the gauze, starting
with the mouth, then the face and finally the rest of the body (Fig. 1a).
A total of 1,519 samples were obtained from anal, oral and skin sites
of infants and mothers at six time points during the first month of
life (1, 3, 7, 14, 21 and 30 d after birth; Supplementary Table 2),
Microbiome composition was characterized by sequencing the V4
region of 16S rRNA gene as previously described12, and 1,016 samples
were used for analysis after quality filtering (see Online Methods).
No adverse events were reported for any of the infants in this study.
Bacterial source-tracking13 of the infant microbiome revealed that
the microbiomes of the four C-section–delivered infants exposed to
vaginal fluids resembled those of vaginally delivered infants, par-
ticularly so during the first week of life (Fig. 1b). The bacterial com-
munity distance between microbiome samples from exposed and
vaginal newborns was lower in anal and skin samples (Fig. 1c). At
day 1, regardless of body site, the microbiomes of babies that were
delivered vaginally or by C-section but exposed to vaginal fluids was
more similar to the maternal vaginal microbiomes than to those of
C-section–delivered (but unexposed) infants (Supplementary Fig. 1).
A progression toward a body-specific configuration was observed
in all of the body sites, either gradually (anus) or quickly (oral and
skin), but both vaginally delivered and exposed C-section–delivered
newborns exhibited a vaginal microbiome–like signature that was
absent in unexposed C-section–delivered babies (Supplementary
Fig. 2). Although variations in microbiome composition between sub-
jects within each of the groups exist (Supplementary Figs. 3–7), we
confirmed the differences between unexposed and exposed C-section–
delivered infants by building a random forest classifier for each body
site14. Samples from unexposed C-section– and vaginally delivered
infants could be classified with high accuracy, confirming that the
mode of delivery shapes the microbial communities of the infants
Partial restoration of the microbiota
of cesarean-born infants via vaginal
Maria G Dominguez-Bello1,2, Kassandra M De Jesus-Laboy2,
Nan Shen3, Laura M Cox1, Amnon Amir4, Antonio Gonzalez4,
Nicholas A Bokulich1, Se Jin Song4,5, Marina Hoashi1,6,
Juana I Rivera-Vinas7, Keimari Mendez7, Rob Knight4,8 &
Jose C Clemente3,9
Exposure of newborns to the maternal vaginal microbiota
is interrupted with cesarean birthing. Babies delivered by
cesarean section (C-section) acquire a microbiota that differs
from that of vaginally delivered infants, and C-section delivery
has been associated with increased risk for immune and
metabolic disorders. Here we conducted a pilot study in which
infants delivered by C-section were exposed to maternal vaginal
fluids at birth. Similarly to vaginally delivered babies, the gut,
oral and skin bacterial communities of these newborns during
the first 30 d of life was enriched in vaginal bacteria—which
were underrepresented in unexposed C-section–delivered
infants—and the microbiome similarity to those of vaginally
delivered infants was greater in oral and skin samples than in
anal samples. Although the long-term health consequences of
restoring the microbiota of C-section–delivered infants remain
unclear, our results demonstrate that vaginal microbes can be
partially restored at birth in C-section–delivered babies.
1School of Medicine, New York University, New York, New York, USA. 2Department of Biology, University of Puerto Rico, Río Piedras Campus, San Juan,
Puerto Rico, USA. 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 4Department of Pediatrics,
University of California San Diego, La Jolla, California, USA. 5Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, USA.
6New York University Tandon School of Engineering, New York, New York, USA. 7Department of Obstetrics and Gynecology, Medical Science Campus, University of
Puerto Rico, San Juan, Puerto Rico, USA. 8Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA.
9Department of Medicine, Division of Clinical Immunology, Icahn School of Medicine at Mount Sinai, New York, New York, USA. Correspondence should be
addressed to M.G.D.-B. (firstname.lastname@example.org) or J.C.C. (email@example.com).
Received 3 July 2015; accepted 22 December 2015; published online 1 February 2016; doi:10.1038/nm.4039
© 2016Nature America, Inc. All rights reserved.
2 nature medicine
(Supplementary Table 3). In relation to C-section–delivered infants
who were not exposed to vaginal fluids, anal, oral and skin samples from
exposed C-section–delivered infants were classified less frequently as
samples from C-section–delivered infants and more often as samples
from vaginally delivered infants (Supplementary Table 3)—with oral
and skin samples being more accurately classified than anal samples.
A random forest classifier built from predicted metagenome content
was equally able to distinguish vaginally delivered and C-section–
delivered infants, with the exposed infants being classified as vaginally
delivered using the oral and skin samples, and as C-section–delivered
from the anal samples, but again less frequently than unexposed
C-section–delivered infants (Supplementar y Table 4).
Microbial colonization of body sites in the newborn occurs quickly,
and changes proceed during the first month in all of the groups. In
anal samples from exposed infants and vaginally delivered infants,
there was an early enrichment of Lactobacillus followed by a bloom
of Bacteroides from week 2, which was not observed in newborns
that were not exposed to vaginal fluids (Fig. 1d and Supplementary
Fig. 2). The anal microbiota of newborns here, as previously reported,
remained distinct from that of adults even at 1 month of life15,16. The
infant skin and oral microbiota, however, acquired a more adult-like
configuration after the first week of life in all three groups. Unexposed
C-section–delivered infants, however, lacked the vaginal bacteria that
were restored by swabbing infants with gauze or that were present in
vaginally delivered infants—particularly anal and skin Lactobacillus
early in life, anal Bacteroides and members of the Bacteroidales family
S24-7 in the skin (Fig. 1d and Supplementary Fig. 2). Maturation of
the infant gut microbiome occurs with the cessation of breastfeeding,
and there are no differences until the fourth month of life between the
microbiomes of infants who are exclusively breast-fed and those from
infants whose diet is supplemented with formula15. Because all of the
infants in our study received breast milk either exclusively or supple-
mented with formula during the first month of life (Supplementary
Table 1), the microbiome composition profiles observed in each
group do not appear to be due to the mode of feeding.
Neonatal bacterial diversity was highest at birth in the anal and oral
sites, and this declined by the third day (Supplementary Fig. 8a,b).
Although this phenomenon is not yet well understood, we have previ-
ously observed it in the gut microbiome of mice17. Although the post-
natal decrease in digestive diversity might reflect the selective effect
of milk on the gut and oral microbiomes, the initially higher diver-
sity could be explained by in utero colonization of the neonate18,19.
The bacterial diversity on the skin of newborns, in contrast, was low-
est at birth and gradually increased during the first month of life
(Supplementary Fig. 8c).
Because C-section–delivered infants were exposed to vaginal flu-
ids through the use of sterile gauzes, we determined how similar the
microbiomes of the gauzes were to those of samples obtained from
the maternal body sites at day 1. We confirmed that the microbiota
of the gauzes that were incubated in the maternal vagina were the
most similar to those of the vaginal samples (Fig. 2a), with both being
enriched for Lactobacillus iners (Supplementary Fig. 9). The bacte-
rial community distance of each gauze sample to its own maternal
vaginal sample was smaller than to those of other mothers, although
Lactobacillus relative abundance (anal)
Lactobacillus relative abundance (skin)
S24 relative abundance (skin)
Bacteroides relative abundance (anal)
0 10 20 300 10 20 30 0 10 20 30
Vaginal Unknown Skin Oral Anal
Vaginal Inoculated C-section
Image: M.J. Schoen
Mouth Face Rest of body
Figure 1 Restoring the
maternal microbiota in
infants born by C-section.
(a) Infants born by
C-section were swabbed
with a gauze that was
incubated in the maternal
vagina 60 min before
the C-section. All mothers
delivering by C-section received
antibiotics (ABX) as part of standard-of-care
treatment (top). The gauze was extracted before the procedure, kept in a sterile (middle) container and used to swab the newborn within the first 1–3
min after birth, starting with the mouth, then the face and the rest of the body (bottom). (b) Proportion of microbiota from anal (top), oral (middle)
and skin (bottom) samples of infants delivered either vaginally (left; n = 7 subjects sampled at six time points), by C-section (unexposed) (right; n = 7
subjects sampled at six time points) or by C-section and exposed to vaginal fluids (middle; n = 4 subjects sampled at six time points) that are estimated
to originate from different maternal sources (colored regions), using bacterial source-tracking. (c) Bacterial community distances (unweighted UniFrac
distances) in anal (left), oral (middle) and skin (right) samples between vaginally delivered babies and C-section–delivered babies that were either
exposed (I-V) or not exposed (C-V) to the vaginal gauze during the first month of life. Because the babies were sampled more frequently during the
first week of life, the x axis is represented as a log scale. Error bars indicate mean ± s.d. *P < 0.01; analysis of variance (ANOVA) and Tukey’s honest
significant difference test. (d) Representative bacterial taxa enriched in infants with perinatal exposure to vaginal fluids during the first month of life.
S24, Bacteroidales family S24-7 members. Error bars indicate mean ± s.d.
© 2016Nature America, Inc. All rights reserved.
nature medicine 3
these differences were not significant (Supplementary Fig. 10).
Power analysis estimated that it would require 35 samples per group to
detect the observed effect size (Cohen’s d = 0.68) with a power of 80%
and a significance level of 0.05. UniFrac distances from the gauzes
to the vaginal samples were significantly smaller than those from
the gauzes to the other body sites (analysis of variance (ANOVA),
P < 0.01; Fig. 2b); bacterial source-tracking further confirmed that
the microbiota of the gauzes is mostly of vaginal origin (Fig. 2c).
Although our sample size was limited and sampling extended
only through the first month after birth, our results suggest that by
exposing the infant to the maternal vaginal microbiota, the bacte-
rial communities of newborns delivered by C-section can be partially
restored to resemble those of vaginally delivered babies. The partial
microbiota restoration provided by the gauze might be due to the
compounded effects of the antibiotic treatments that accompany
the C-section procedure and the suboptimal bacterial transfer from
the vagina to the gauze and then to the baby. However, there was no
apparent clustering of the vaginal microbiota on the basis of exposure to
antibiotics (Fig. 2a; arrows indicate mothers unexposed to antibiotics).
Bacterial diversity was not lower in the vaginal microbiota of mothers
who received antibiotics (Fig. 2d), and no clear differences were
observed in taxonomic composition either (Fig. 2e). The abundance of
Lactobacillus was not depleted in the vaginal microbiota of antibiotic-
exposed mothers as compared to that of unexposed mothers (Student’s
t-test, P = 0.618). Both exposed and unexposed C-section–delivered
infants were comparable in terms of antibiotic exposure and feeding
(Supplementary Table 1), suggesting that the differences observed
in microbiome composition between these two groups can be most
parsimoniously explained by the exposure to the vaginally swabbed
gauze. A larger sample size will be needed to clarify the effect of peri-
natal antibiotics on the vaginal microbiome and, subsequently, on the
microbiome of the gauze that was incubated in the maternal vagina
and on the bacterial load received by the infant. Determining more
effective approaches to transfer the maternal microbiota to newborns
and, more importantly, establishing which keystone species newborn
infants should acquire at birth, are important to replicating the benefi-
cial effects provided by vaginal delivery in C-section–delivered infants.
The partial microbial restoration observed in our study could be due
to the fact that infants are exposed a single time to topical application
of vaginal fluids. Furthermore, particular body sites (such as mouth
and skin) were more amenable to inoculation than others. A modified
protocol that exposes newborns repeatedly to the gauze would more
faithfully reproduce the extended exposure that vaginally delivered
infants receive and might potentially improve microbial restoration,
although this hypothesis remains to be tested. Enteral administration
of key bacterial species could further supplement the method described
here; however, extensive research would be required to ensure the safety
and efficacy of such an approach. We stress that our work represents
a proof of principle on a small cohort and with limited follow-up over
time. Labor is a complex process that cannot be fully recaptured by our
procedure, and which encompasses multiple factors beyond the mere
transmission of microbes from mother to infant. Finally, extended
longitudinal analysis of larger cohorts is needed to determine whether
this procedure has any effects on diseases later in life.
Methods and any associated references are available in the online
version of the paper.
Accession codes. European Bioinformatics Institute (EBI): sequence data
have been deposited under study accession number PRJEB10914.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
This work was partially supported by the C&D Research Fund (M.G.D.-B.),
the US National Institutes of Health grant no. R01 DK090989 (M.G.D.-B),
the Crohn’s and Colitis Foundation of America grant no. 362048 (J.C.C.) and the
Sinai Ulcerative Colitis: Clinical, Experimental & Systems Studies philanthropic
grant (J.C.C.). Sequencing at the New York University Genome Technology
Center was partially supported by the Cancer Center Support grant no.
P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. Computing
was partially supported by the Department of Scientific Computing at the
Icahn School of Medicine at Mount Sinai. We acknowledge the contribution of
the students who participated in obtaining the samples and the metadata: S.M.
Rodriguez, J.F. Ruiz, N. Garcia and J.L. Rivera-Correa. We also thank M.J. Blaser
Bacterial diversity (PD)
** ** **
13 14 17 18
PC2 (9%) Anal
a b c d e
No antibiotics Antibiotics
Figure 2 Transmission of maternal vaginal microbes to the gauze. (a) Principal coordinate (PC) analysis
of unweighted UniFrac community distances for maternal anal (red), oral (green), skin (purple) and vaginal
(yellow) microbiota (n = 95 samples) and gauze (blue) (n = 4) microbiota at day 1. Vaginal gauze bacteria resemble
vaginal communities. Arrows indicate vaginal samples from mothers unexposed to antibiotics. (b) Bacterial community
distances between gauzes and each maternal body site at day 1. Error bars indicate mean ± s.d. **P < 0.001; ANOVA and
Tukey’s honest significant difference test. (c) Proportion of gauze sample microbiota estimated to originate from different
maternal sources using bacterial source-tracking. Each stacked bar represents a gauze sample from a different mother.
Oral microbiota were not found to be a potential source of the bacterial communities for any gauze and are not indicated
in the legend. (d) Box plot of bacterial diversity (Faith’s phylogenetic diversity; PD) of maternal vaginal microbiota in mothers that received (n = 13) or
did not receive (n = 5) antibiotics before vaginal sampling before delivery. Top and bottom of the boxes indicate the first and third quartile, respectively.
The upper (or lower) whisker extends from the top of the box to the highest (or lowest) value within 1.5 times the inter-quartile range, defined as the
distance between the first and third quartiles. n.s., not significant; two-tailed Student’s t-test. (e) Relative abundance of bacterial genera in the vaginal
microbiota of mothers that received (n = 13) or did not receive (n = 5) antibiotics before vaginal sampling before delivery. Genera that could not be fully
resolved are replaced by the taxonomic family to which they belong. The number below each bar indicates the sample identity.
© 2016Nature America, Inc. All rights reserved.
4 nature medicine
for discussions and critical comments, and three anonymous reviewers for their
suggestions to improve this manuscript.
M.G.D.-B. designed the study. M.G.D.-B., K.M.D.J.-L., J.I.R.-V. and K.M. collected
and processed specimens. M.G.D.-B. sequenced and generated data. N.S., L.M.C.,
A.A., A.G., N.A.B., S.J.S., M.H. and J.C.C. performed experiments. M.G.D.-B., N.S.,
L.M.C., A.A., A.G., N.A.B., S.J.S., M.H., R.K. and J.C.C. analyzed data. M.G.D.-B.
and J.C.C. drafted the manuscript. All authors reviewed the final manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Reprints and permissions information is available online at http://www.nature.com/
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© 2016Nature America, Inc. All rights reserved.
Study design. The inclusion criteria for mothers participating in this study
were healthy mothers, as assessed by their doctors who were delivering either
vaginally or by scheduled C-section. Mothers scheduled to have a C-section
were offered the opportunity to participate in the study and were divided into
the two groups based on their willingness to have their newborns swabbed with
the gauze. For the group of C-section–delivered infants exposed to maternal
vaginal fluids, mothers had to have negative results for the standard-of-care
tests of sexually transmitted diseases (STDs)—including HIV, Chlamydia
and group B Streptococcus (GBS, standard test at 35 weeks by culturing)—no
signs of vaginosis or viral infections as determined by their obstetrician and a
vaginal pH < 4.5 at 1–2 h preceding the procedure. Of the 14 mothers whose
infants were not exposed to the gauze (7 vaginal and 7 C-section deliveries),
three were GBS positive, and of which two were delivered by C-section and
one was by vaginal delivery (Supplementary Table 1). All mothers received
standard-of-care treatment, including preventive perinatal antibiotics (Beta
lactams: mostly cephalosporins or penicillin; see Supplementary Table 1 for
details) for mothers who underwent cesarean section or for vaginally delivering
GBS-positive mothers. The study was approved by the Institutional Review
Boards from the University of Puerto Rico, Medical Sciences Campus
(A9710112) and from the Rio Piedras (1011-107) campus. All C-sections in
this study were due to previous C-sections, and the procedure was conducted at
the University Adult’s Hospital, Puerto Rico Medical Center. Written informed
consent was obtained from all participants.
Microbial restoration procedure. Within the hour prior to the procedure,
maternal vaginal pH was measured using a sterile swab and a paper pH strip
(Fisher). Once the pH was confirmed to be <4.5, an 8-cm × 8-cm four-layered
gauze (Fisherbrand cat# 22028558) was folded like a fan, and then in half, wet
with sterile saline solution and inserted in the vagina for 1 h. Right before the
C-section surgery started, the gauze was extracted and placed in a sterile col-
lector and kept at room temperature. As soon as the baby was brought to the
neonate lamp and within 1 min after delivery, the infant was swabbed with the
gauze, starting on the lips, followed by the face, thorax, arms, legs, genitals and
anal region, and finally the back. The swabbing took approximately 15 s. The
neonatologist then proceeded to perform the standard detailed examination
of the newborn.
Sample collection and processing. Sampling with sterile swabs in different
body sites took place within the first 5 min after birth in all babies (including
the vaginal gauze–exposed C-section group, who were sampled after the gauze
swabbing procedure), then at day 3 and then weekly for the first month. Sampled
body sites included oral mucosa, forehead, right arm, right foot and anal region
of the baby, and the same sites of the mother plus a vaginal swab (Supplementary
Table 2). Gauze samples were obtained from a 1-cm2 area from the center of
the gauze. All samples were transported to the laboratory with ice packs within
2 h after collection and stored at −80 °C until further processing. DNA was
extracted from samples using the MoBio Powersoil Kit according to the manu-
facturer’s instructions, modified as described in the Earth Microbiome Project
Sequencing and data processing. Sequencing of the swabs and gauzes was
performed at the NYU Genome Technology Center using the Illumina MiSeq
sequencing instrument, with v2 reagents and 2 × 250 cartridge. Raw reads
were de-multiplexed and quality-filtered using QIIME v1.8.0 with default
parameters20. Quality-filtered reads were clustered into operational taxonomic
units (OTUs) using an open-reference algorithm21 and Greengenes v13_8 as
a reference set22. Samples that had at least 1,000 sequences (n = 1,016) were
further analyzed, resulting in a total of 6,515,724 sequences (mean 6,413 ±
4,593; median 5,360 sequences). Alpha diversity on rarefied tables was esti-
mated by using Faith’s phylogenetic diversity23 and beta diversity by using
Bacterial source-tracking. To estimate the sources of the microbial commu-
nities observed in each of the three infant groups at different body sites and
time points, we used SourceTracker (v1.0), a Bayesian approach for bacterial
source-tracking13. Samples from each body site in the infants were designated
as sinks, and samples from all of the body sites of the corresponding mother
were tagged as sources. Source-tracking of bacterial communities in the gauzes
were performed similarly, designating gauzes as sinks and paired maternal body
sites as potential sources.
Supervised learning classification. A random forest classifier was built for
each body site using 500 trees and a leave-one-out error model as previously
described14. To account for subsampling variability, the input tables were rarefied
ten times and results averaged over the total. The confusion matrices represent
the mean and s.d. percentage of samples from the true class assigned to each of
the possible classes (vaginal, exposed, C-section) for each body site.
Predicted metagenome. OTU tables were filtered to remove de novo OTUs
(i.e., not found in Greengenes). Metagenomic content was estimated using
PICRUSt25, first normalizing each OTU in the filtered tables by its correspond-
ing copy number and then predicting the abundance of functional traits from
the normalized OTU counts.
Deblurring. To identify with higher resolution the Lactobacillus present in
the samples we used deblurring, a novel denoising method for Illumina-based
amplicon sequencing. By using the raw reads as input, deblurring processes each
sample independently and tries to remove all sequences derived from sequenc-
ing-read errors or PCR errors, based on an upper bound on error probabilities.
Briefly, the deblurring algorithm steps and parameters used for the current
study are the following. (i) Trim the all reads to constant length (150 bp) and de-
replicate, retaining the total number of reads per unique sequence. (ii) Subsample
to 4,000 reads (per sample). (iii) Discard all singleton reads. (iv) Remove
sequences containing known sequencing artifacts (PhiS or adaptor sequences).
(v) Perform multiple-sequence alignment using MAFFT v7.130b26. (vi) Perform
the actual deblurring: iterate on all sequences from highest frequency to lowest:
(a) for each sequence, reduce the frequency of neighboring sequences (based
on Hamming distance + indel (insertion and deletion)) according to the
distance dependent maximal error profile—the error profile used ranges from
6% maximal read error for Hamming distance 1, 2% for distance 2, and down
to 0.1% for Hamming distance 10; and (b) if the resulting frequency is lower
than 0, then remove the sequence from the list. (vii) Remove chimeras from the
resulting sequences using de novo chimera detection with usearch 5.2.236 and
the parameter ‘uchime_denovo’.
The performance of deblurring has been validated in simulations and mock
mixtures, and deblurring was shown to retain the exact actual sequences in the
sample while removing most of the sequencing error– and PCR error–derived
sequences, with the ability to detect sequences differing by as little as one nucle-
otide over the entire sequenced region. Additional details on deblurring, and the
source code, can be found at https://github.com/biocore/deblur.
Statistical analyses. All technicians processing the samples were blinded to the
group allocations. Computational analyses were not blinded owing to the use of
supervised learning methods, which require knowledge of the groups. Because
our study was designed as a proof of concept, sample size was not estimated
a priori. Statistical tests were performed using QIIME 1.8.0 with default
parameters and R 3.2.2.
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