April 29, 2020 |
Asset Tag: Amplicon
April 21, 2020 |
a-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY.
Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent a-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.
October 23, 2019 |
Dynamics of coral-associated microbiomes during a thermal bleaching event.
Coral-associated microorganisms play an important role in their host fitness and survival. A number of studies have demonstrated connections between thermal tolerance in corals and the type/relative abundance of Symbiodinium they harbor. More recently, the shifts in coral-associated bacterial profiles were also shown to be linked to the patterns of coral heat tolerance. Here, we investigated the dynamics of Porites lutea-associated bacterial and algal communities throughout a natural bleaching event, using full-length 16S rRNA and internal transcribed spacer sequences (ITS) obtained from PacBio circular consensus sequencing. We provided evidence of significant changes in the structure and diversity of coral-associated microbiomes during thermal stress. The balance of the symbiosis shifted from a predominant association between corals and Gammaproteobacteria to a predominance of Alphaproteobacteria and to a lesser extent Betaproteobacteria following the bleaching event. On the contrary, the composition and diversity of Symbiodinium communities remained unaltered throughout the bleaching event. It appears that the switching and/or shuffling of Symbiodinium types may not be the primary mechanism used by P. lutea to cope with increasing seawater temperature. The shifts in the structure and diversity of associated bacterial communities may contribute more to the survival of the coral holobiont under heat stress.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
October 23, 2019 |
High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.
Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.
July 19, 2019 |
Polymerase specific error rates and profiles identified by single molecule sequencing.
DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases. Copyright © 2016 Elsevier B.V. All rights reserved.
July 7, 2019 |
ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing.
The characterization of the ABO blood group status is vital for blood transfusion and solid organ transplantation. Several methods for the molecular characterization of the ABO gene, which encodes the alleles that give rise to the different ABO blood groups, have been described. However, the application of those methods has so far been restricted to selected samples and not been applied to population-scale analysis.We describe a cost-effective method for high-throughput genotyping of the ABO system by next generation sequencing. Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. We report the allele group frequencies calculated on a subset of more than 110,000 sampled individuals of German origin. Further we discuss the potential of the workflow for high resolution genotyping taking the observed allele group frequencies into account. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.The described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.
July 7, 2019 |
RIFRAF: a frame-resolving consensus algorithm.
Protein coding genes can be studied using long-read next generation sequencing. However, high rates of indel sequencing errors are problematic, corrupting the reading frame. Even the consensus of multiple independent sequence reads retains indel errors. To solve this problem, we introduce Reference-Informed Frame-Resolving multiple-Alignment Free template inference algorithm (RIFRAF), a sequence consensus algorithm that takes a set of error-prone reads and a reference sequence and infers an accurate in-frame consensus. RIFRAF uses a novel structure, analogous to a two-layer hidden Markov model: the consensus is optimized to maximize alignment scores with both the set of noisy reads and with a reference. The template-to-reads component of the model encodes the preponderance of indels, and is sensitive to the per-base quality scores, giving greater weight to more accurate bases. The reference-to-template component of the model penalizes frame-destroying indels. A local search algorithm proceeds in stages to find the best consensus sequence for both objectives.Using Pacific Biosciences SMRT sequences from an HIV-1 env clone, NL4-3, we compare our approach to other consensus and frame correction methods. RIFRAF consistently finds a consensus sequence that is more accurate and in-frame, especially with small numbers of reads. It was able to perfectly reconstruct over 80% of consensus sequences from as few as three reads, whereas the best alternative required twice as many. RIFRAF is able to achieve these results and keep the consensus in-frame even with a distantly related reference sequence. Moreover, unlike other frame correction methods, RIFRAF can detect and keep true indels while removing erroneous ones.RIFRAF is implemented in Julia, and source code is publicly available at https://github.com/MurrellGroup/Rifraf.jl.Supplementary data are available at Bioinformatics online.