Targeted Sequencing and Custom Primers with MRDNA

9. Mol Ecol Resour. 2013 Jul;13(4):620-33. doi: 10.1111/1755-0998.12103. Epub 2013

Apr 17.


Quantifying sequence proportions in a DNA-based diet study using Ion Torrent

amplicon sequencing: which counts count?


Deagle BE(1), Thomas AC, Shaffer AK, Trites AW, Jarman SN.


Author information:

(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.


DOI: 10.1111/1755-0998.12103

PMID: 23590207  [PubMed - indexed for MEDLINE]



10. Eur J Dent. 2015 Jan-Mar;9(1):127-32. doi: 10.4103/1305-7456.149661.


16S rRNA gene-based metagenomic analysis identifies a novel bacterial

co-prevalence pattern in dental caries.


Jagathrakshakan SN(1), Sethumadhava RJ(1), Mehta DT(2), Ramanathan A(2).


Author information:

(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.


DOI: 10.4103/1305-7456.149661

PMCID: PMC4319289

PMID: 25713496  [PubMed]



11. J Microbiol Methods. 2015 Jan;108:103-11. doi: 10.1016/j.mimet.2014.11.013. Epub

2014 Dec 2.


Comparative assessment of next-generation sequencing, denaturing gradient gel

electrophoresis, clonal restriction fragment length polymorphism and

cloning-sequencing as methods for characterizing commercial microbial consortia.


Samarajeewa AD(1), Hammad A(2), Masson L(3), Khan IU(4), Scroggins R(2),

Beaudette LA(2).


Author information:

(1)Biological Assessment and Standardization Section, Environment Canada, 335

River Road, Ottawa, Ontario, Canada K1V 1C7. Electronic address: (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.


DOI: 10.1016/j.mimet.2014.11.013

PMID: 25479430  [PubMed - indexed for MEDLINE]



12. MBio. 2014 Sep 30;5(5):e01548-14. doi: 10.1128/mBio.01548-14.


Intestinal dysbiosis associated with systemic lupus erythematosus.


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).


Author information:

(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.


DOI: 10.1128/mBio.01548-14

PMCID: PMC4196225

PMID: 25271284  [PubMed - indexed for MEDLINE]

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