A northern glacial refugium for bank voles (Clethrionomys glareolus).

peer reviewedThere is controversy and uncertainty on how far north there were glacial refugia for temperate species during the Pleistocene glaciations and in the extent of the contribution of such refugia to present-day populations. We examined these issues using phylogeographic analysis of a European woodland mammal, the bank vole (Clethrionomys glareolus). A Bayesian coalescence analysis indicates that a bank vole population survived the height of the last glaciation (≈25,000–10,000 years B.P.) in the vicinity of the Carpathians, a major central European mountain chain well north of the Mediterranean areas typically regarded as glacial refugia for temperate species. Parameter estimates from the fitted isolation with migration model show that the divergence of the Carpathian population started at least 22,000 years ago, and it was likely followed by only negligible immigration from adjacent regions, suggesting the persistence of bank voles in the Carpathians through the height of the last glaciation. On the contrary, there is clear evidence for gene flow out of the Carpathians, demonstrating the contribution of the Carpathian population to the colonization of Europe after the Pleistocene. These findings are consistent with data from animal and plant fossils recovered in the Carpathians and provide the clearest phylogeographic evidence to date of a northern glacial refugium for temperate species in Europe

Characterization of Bacteria in Biopsies of Colon and Stools by High Throughput Sequencing of the V2 Region of Bacterial 16S rRNA Gene in Human

BACKGROUND: The characterization of the human intestinal microflora and their interactions with the host have been identified as key components in the study of intestinal disorders such as inflammatory bowel diseases. High-throughput sequencing has enabled culture-independent studies to deeply analyze bacteria in the gut. It is possible with this technology to systematically analyze links between microbes and the genetic constitution of the host, such as DNA polymorphisms and methylation, and gene expression. METHODS AND FINDINGS: In this study the V2 region of the bacterial 16S ribosomal RNA (rRNA) gene using 454 pyrosequencing from seven anatomic regions of human colon and two types of stool specimens were analyzed. The study examined the number of reads needed to ascertain differences between samples, the effect of DNA extraction procedures and PCR reproducibility, and differences between biopsies and stools in order to design a large scale systematic analysis of gut microbes. It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances. Distances between samples became stable after 1,000 reads. (2) Difference of extracted bacteria was observed between the two DNA extraction methods. In particular, Firmicutes Bacilli were not extracted well by one method. (3) Quantitative and qualitative difference in bacteria from ileum to rectum colon were not observed, but there was a significant positive trend between distances within colon and quantitative differences. Between sample type, biopsies or stools, quantitative and qualitative differences were observed. CONCLUSIONS: Results of human colonic bacteria analyzed using high-throughput sequencing were highly dependent on the experimental design, especially the number of sequence reads, DNA extraction method, and sample type

Comparative phylogeography of forest rodents (Apodemus sylvaticus, A. flavicollis and Clethrionomys glareolus) in the western Palearctic region: how the same quaternary climatic changes led to different evolutionary histories

peer reviewe

Characterization of bacteria in biopsies of colon and stools by high throughput sequencing of the V2 region of bacterial 16S rRNA gene in human.

BackgroundThe characterization of the human intestinal microflora and their interactions with the host have been identified as key components in the study of intestinal disorders such as inflammatory bowel diseases. High-throughput sequencing has enabled culture-independent studies to deeply analyze bacteria in the gut. It is possible with this technology to systematically analyze links between microbes and the genetic constitution of the host, such as DNA polymorphisms and methylation, and gene expression.Methods and findingsIn this study the V2 region of the bacterial 16S ribosomal RNA (rRNA) gene using 454 pyrosequencing from seven anatomic regions of human colon and two types of stool specimens were analyzed. The study examined the number of reads needed to ascertain differences between samples, the effect of DNA extraction procedures and PCR reproducibility, and differences between biopsies and stools in order to design a large scale systematic analysis of gut microbes. It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances. Distances between samples became stable after 1,000 reads. (2) Difference of extracted bacteria was observed between the two DNA extraction methods. In particular, Firmicutes Bacilli were not extracted well by one method. (3) Quantitative and qualitative difference in bacteria from ileum to rectum colon were not observed, but there was a significant positive trend between distances within colon and quantitative differences. Between sample type, biopsies or stools, quantitative and qualitative differences were observed.ConclusionsResults of human colonic bacteria analyzed using high-throughput sequencing were highly dependent on the experimental design, especially the number of sequence reads, DNA extraction method, and sample type

Association between coverage and the number of OTUs.

<p>(A) The number of OTUs sampled as a function of number of reads. The data points represent mean ± SD of five randomized samplings. (B) Coverage to detect OTU with different frequencies with ≥95% of confidence. The data points were estimated based on the binomial distribution (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016952#s4" target="_blank">Materials and Methods</a>). The x axis is shown in logarithmic scale.</p

Association between coverage and UniFrac distances at different coverage.

<p>(A) Weighted and (B) unweighted UniFrac distances. For ‘Between individuals’ each data point includes 36 distances calculated from 6 samples of individual B and 6 samples of individual C. For ‘Within same individual’ each data point includes 18 distances (9 within sample B and 9 within sample C). Mean ± SD.</p

UniFrac distances between different PCR conditions.

<p>(A) Weighted and (B) unweighted UniFrac distances. UniFrac distances were calculated between the most used PCR condition (annealing temperature: 55°C and the number of cycle: 25 cycles) and three PCR conditions; the same PCR condition (technical replication), the PCR condition of more cycles (55°C and 35 cycles), and the PCR condition of lower annealing temperature (50°C and 25 cycles). * p<0.05, ** p<0.01</p

UniFrac distances between sequencing methods.

<p>(A) Weighted and (B) unweighted UniFrac distances. Reads by 454 Standard chemistry were compared with technical replicates of the same DNAs sequenced by 454 Standard chemistry (Standard), with 454 Titanium chemistry (no trim), and with 454 Titanium chemistry with sequences trimmed at 270 bp (trim). ** p<0.01, **** p<0.0001</p