9. Mol Ecol Resour. 2013 Jul;13(4):620-33. doi: 10.1111/1755-0998.12103. Epub 2013
Deagle BE(1), Thomas AC, Shaffer AK, Trites AW, Jarman SN.
(1)Australian Antarctic Division, Channel Highway, Kingston, Tas, Australia.
A goal of many environmental DNA barcoding studies is to infer quantitative
information about relative abundances of different taxa based on sequence read
proportions generated by high-throughput sequencing. However, potential biases
associated with this approach are only beginning to be examined. We sequenced DNA
amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to
investigate whether sequence counts could be used to quantify the seals' diet.
Seals were fed fish in fixed proportions, a chordate-specific mitochondrial 16S
marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent
PGM™. For a given set of bioinformatic parameters, there was generally low
variability between scat samples in proportions of prey species sequences
recovered. However, proportions varied substantially depending on sequencing
direction, level of quality filtering (due to differences in sequence quality
between species) and minimum read length considered. Short primer tags used to
identify individual samples also influenced species proportions. In addition,
there were complex interactions between factors; for example, the effect of
quality filtering was influenced by the primer tag and sequencing direction.
Resequencing of a subset of samples revealed some, but not all, biases were
consistent between runs. Less stringent data filtering (based on quality scores
or read length) generally produced more consistent proportional data, but overall
proportions of sequences were very different than dietary mass proportions,
indicating additional technical or biological biases are present. Our findings
highlight that quantitative interpretations of sequence proportions generated via
high-throughput sequencing will require careful experimental design and
thoughtful data analysis.
© 2013 John Wiley & Sons Ltd.
PMID: 23590207 [PubMed - indexed for MEDLINE]
10. Eur J Dent. 2015 Jan-Mar;9(1):127-32. doi: 10.4103/1305-7456.149661.
Jagathrakshakan SN(1), Sethumadhava RJ(1), Mehta DT(2), Ramanathan A(2).
(1)Department of Prosthodontia, Sree Balaji Dental College and Hospital, Bharath
University, Narayanapuram, Pallikaranai, Chennai, Tamil Nadu, India.
(2)Department of Human Genetics Laboratory, Central Research Facility, Sree
Balaji Medical and Dental College and Hospital, Bharath University,
Narayanapuram, Pallikaranai, Chennai, Tamil Nadu, India.
OBJECTIVE: To identify the prevalence of acidogenic and nonacidogenic bacteria in
patients with polycaries lesions, and to ascertain caries specific bacterial
prevalence in relation to noncaries controls.
MATERIALS AND METHODS: Total genomic DNA extracted from saliva of three adults
and four children from the same family were subjected to 16S rRNA gene sequencing
analysis on a next generation sequencer, the PGS-Ion Torrent. Those bacterial
genera with read counts > 1000 were considered as significant in each of the
subject and used to associate the occurrence with caries.
RESULTS AND CONCLUSION: Sequencing analysis indicated a higher prevalence of
Streptococcus, Rothia, Granulicatella, Gemella, Actinomyces, Selenomonas,
Haemophilus and Veillonella in the caries group relative to controls. While
higher prevalence of Streptococcus, Rothia and Granulicatella were observed in
all caries samples, the prevalence of others was observable in 29-57% of samples.
Interestingly, Rothia and Selenomonas, which are known to occur within anaerobic
environments of dentinal caries and subgingival plaque biofilms, were seen in the
saliva of these caries patients. Taken together, the study has identified for the
first time a unique co-prevalence pattern of bacteria in caries patients that may
be explored as distinct caries specific bacterial signature to predict
cariogenesis in high-risk primary and mixed dentition age groups.
PMID: 25713496 [PubMed]
11. J Microbiol Methods. 2015 Jan;108:103-11. doi: 10.1016/j.mimet.2014.11.013. Epub
2014 Dec 2.
Samarajeewa AD(1), Hammad A(2), Masson L(3), Khan IU(4), Scroggins R(2),
(1)Biological Assessment and Standardization Section, Environment Canada, 335
River Road, Ottawa, Ontario, Canada K1V 1C7. Electronic address:
email@example.com. (2)Biological Assessment and Standardization
Section, Environment Canada, 335 River Road, Ottawa, Ontario, Canada K1V 1C7.
(3)National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Quebec,
Canada H4P 2R2. (4)Eastern Cereal and Oilseed Research Centre (ECORC),
Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, Canada K1A
Characterization of commercial microbial consortia products for human and
environmental health risk assessment is a major challenge for regulatory
agencies. As a means to develop an approach to assess the potential environmental
risk of these products, research was conducted to compare four genomics methods
for characterizing bacterial communities; (i) Denaturing Gradient Gel
Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism
(C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger
sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent
technology. A commercially available microbial consortium, marketed as a
remediation agent for degrading petroleum hydrocarbon contamination in soil and
water, was assessed. The bacterial composition of the commercial microbial
product was characterized using the above four methods. PCR amplification of 16S
rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS
and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a
promising tool for initial screening by detecting the majority of bacteria in the
consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion
Torrent sequencing detected some of the bacteria that were claimed to be in the
product, while three other methods failed to detect these specific bacteria.
However, the relative proportions of the microbial composition detected by Ion
Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave
comparable results across these three methods. The discrepancy of the Ion Torrent
results may be due to the short read length generated by this technique and the
targeting of different variable regions on the 16S rRNA gene used in this study.
Arcobacter spp. a potential pathogenic bacteria was detected in the product by
all methods, which was further confirmed using genus and species-specific PCR,
RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp.
was not confirmed. This study suggests that a combination of two or more methods
may be required to ascertain the microbial constituents of a commercial microbial
consortium reliably and for the presence of potentially human pathogenic
Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.
PMID: 25479430 [PubMed - indexed for MEDLINE]
12. MBio. 2014 Sep 30;5(5):e01548-14. doi: 10.1128/mBio.01548-14.
Hevia A(1), Milani C(2), López P(3), Cuervo A(4), Arboleya S(1), Duranti S(2),
Turroni F(2), González S(4), Suárez A(3), Gueimonde M(1), Ventura M(2), Sánchez
B(5), Margolles A(5).
(1)Instituto de Productos Lácteos de Asturias, Consejo Superior de
Investigaciones Científicas, Villaviciosa, Asturias, Spain. (2)Laboratory of
Probiogenomics, Department of Life Sciences, University of Parma, Parma, Italy.
(3)Immunology Area, Department of Functional Biology, University of Oviedo,
Asturias, Spain. (4)Physiology Area, Department of Functional Biology, University
of Oviedo, Asturias, Spain. (5)Instituto de Productos Lácteos de Asturias,
Consejo Superior de Investigaciones Científicas, Villaviciosa, Asturias, Spain
Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune
disease in humans and is characterized by the presence of hyperactive immune
cells and aberrant antibody responses to nuclear and cytoplasmic antigens,
including characteristic anti-double-stranded DNA antibodies. We performed a
cross-sectional study in order to determine if an SLE-associated gut dysbiosis
exists in patients without active disease. A group of 20 SLE patients in
remission, for which there was strict inclusion and exclusion criteria, was
recruited, and we used an optimized Ion Torrent 16S rRNA gene-based analysis
protocol to decipher the fecal microbial profiles of these patients and compare
them with those of 20 age- and sex-matched healthy control subjects. We found
diversity to be comparable based on Shannon's index. However, we saw a
significantly lower Firmicutes/Bacteroidetes ratio in SLE individuals (median
ratio, 1.97) than in healthy subjects (median ratio, 4.86; P < 0.002). A lower
Firmicutes/Bacteroidetes ratio in SLE individuals was corroborated by
quantitative PCR analysis. Notably, a decrease of some Firmicutes families was
also detected. This dysbiosis is reflected, based on in silico functional
inference, in an overrepresentation of oxidative phosphorylation and glycan
utilization pathways in SLE patient microbiota.IMPORTANCE: Growing evidence
suggests that the gut microbiota might impact symptoms and progression of some
autoimmune diseases. However, how and why this microbial community influences SLE
remains to be elucidated. This is the first report describing an SLE-associated
intestinal dysbiosis, and it contributes to the understanding of the interplay
between the intestinal microbiota and the host in autoimmune disorders.
Copyright © 2014 Hevia et al.
PMID: 25271284 [PubMed - indexed for MEDLINE]