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April 21, 2020

Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low owing to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming commonly used primers. Based on comparing the resulting long 16S rRNA gene fragments with public databases from all habitats, we found several novel family- to phylum-level archaeal taxa from topsoil and surface water. Our results suggest that archaeal diversity has been largely overlooked due to the limitations of available primers, and that improved primer pairs enable to estimate archaeal diversity more accurately. © 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Endogenous pararetrovirus sequences are widely present in Citrinae genomes.

Endogenous pararetroviruses (EPRVs) are characterized in several plant genomes and their biological effects have been reported. In this study, hundreds of EPRV segments were identified in six Citrinae genomes. A total of 1034 EPRV segments were identified in the genomes of sweet orange, 2036 in pummelo, 598 in clementine mandarin, 752 in Ichang papeda, 2060 in citron and 245 in atalantia. Genomic analysis indicated that EPRV segments tend to cluster as hot spots in the genomes, particularly on chromosome 2 and 5. Large numbers of simple repeats and transposable elements were identified in the 2-kb flanking regions of the EPRV segments. Comparative genomic analysis and PCR experiments showed that there are highly conserved EPRV segments and species-specific EPRV segments between the Citrinae genomes. Phylogenetic analysis suggested that the integration events of EPRVs could initiate in a common progenitor of Citrinae species and repeatedly occur during the Citrinae divergence.Copyright © 2018 Elsevier B.V. All rights reserved.

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April 21, 2020

Potential of TLR-gene diversity in Czech indigenous cattle for resistance breeding as revealed by hybrid sequencing

A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3–5×?coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60× per gene). The diversity in conserved CRP and CR was similar to the diversity in conserved and modern CRP, representing 76.4?% and 70.9?% of its variants, respectively. Sixty-eight (54.4?%) polymorphisms in the five TLR genes were shared by the two breeds, whereas 38 (30.4?%) were specific to the production herd of CRP; 4 (3.2?%) were specific to the broad CRP population; 7 (5.6?%) were present in both conserved populations; 5 (4.0?%) were present solely for the conserved CRP; and 3 (2.4?%) were restricted to CR. Consequently, gene pool erosion related to intensive breeding did not occur in Czech Simmental cattle. Similarly, no considerable consequences were found from known bottlenecks in the history of Czech Red cattle. On the other hand, the distinctness of the conserved populations and their potential for resistance breeding were only moderate. This relationship might be transferable to other non-abundant historical cattle breeds that are conserved as genetic resources. The estimates of polymorphism impact using Variant Effect Predictor and SIFT software tools allowed for the identification of candidate single-nucleotide polymorphisms (SNPs) for association studies related to infection resistance and targeted breeding. Knowledge of TLR-gene diversity present in Czech Simmental populations may aid in the potential transfer of variant characteristics from other breeds.

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April 21, 2020

Next generation sequencing characterizes HLA diversity in a registry population from the Netherlands.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 assignments of 1009 unrelated volunteers for the unrelated donor registry in The Netherlands. The analysis characterizes all HLA exons and introns for class I alleles; at least exons 2 to 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 229 class I and 71 class II; 36 of these alleles are novel. The majority (approximately 98%) of the cumulative allele frequency at each locus is contributed by alleles that appear three or more times. Alleles encoding protein variation outside of the antigen recognition domains are 0.6% of the class I assignments and 5.3% of the class II assignments. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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April 21, 2020

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life.

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.

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April 21, 2020

The role of long-term mineral and organic fertilisation treatment in changing pathogen and symbiont community composition in soil

Application of organic fertilisers to soil prevents erosion, improves fertility and may suppress certain soil-borne plant pathogens, but it is still unclear how different trophic groups of fungi and oomycetes respond to long-term fertilisation treatment. The objective of the study was to examine the effect of different fertilisation regimes on fungal and oomycete pathogen- and mycorrhizal symbiont diversity and community structure in both soil and roots, using PacBio SMRT sequencing. The field experiment included three fertilisation treatments that have been applied since 1989: nitrogen fertilisation (WOM), nitrogen fertilisation with manure amendment (FYM) and alternative organic fertilisation (AOF), each applied at five different rates. Soil samples were collected three times during the growing season, while root samples were collected during the flowering stage. There was no influence of the studied variables on soil and root pathogen richness. Contrary to our hypothesis, pathogen relative abundance in both soil and roots was significantly higher in plots with the AOF treatment. Furthermore, richness and relative abundance of arbuscular mycorrhizal (AM) fungi decreased significantly in the AOF treatment. Permutational analysis of variance (PERMANOVA) demonstrated the effect of fertilisation treatment on pathogen community composition in both soil and roots. Our findings indicate that organic fertilisers may not always benefit soil microbial community composition. Therefore, further studies are needed to understand how fertilisation affects mycorrhizal mutualists and pathogens.

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April 21, 2020

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

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April 21, 2020

Patterns of non-ARD variation in more than 300 full-length HLA-DPB1 alleles.

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation. The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A???G variant, a known expression marker located in the 3′-UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6?kb stretch at the 3′-end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles. The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Recipients receiving better HLA-matched hematopoietic cell transplantation grafts, uncovered by a novel HLA typing method, have superior survival: A retrospective study

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplanta- tion (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technolo- gies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity out- side of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were ret- rospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next- generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typ- ing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P= .022). Survival was also significantly better in 12/12 UHR HLA- matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% ver- sus 40.1%, P= .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

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April 21, 2020

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

An Annotated Genome for Haliotis rufescens (Red Abalone) and Resequenced Green, Pink, Pinto, Black, and White Abalone Species.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498?Gb; the N50 was 1.895?Mb, and the longest scaffold was 13.2?Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255?bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.

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April 21, 2020

Nephromyces encodes a urate metabolism pathway and predicted peroxisomes, demonstrating that these are not ancient losses of apicomplexans.

The phylum Apicomplexa is a quintessentially parasitic lineage, whose members infect a broad range of animals. One exception to this may be the apicomplexan genus Nephromyces, which has been described as having a mutualistic relationship with its host. Here we analyze transcriptome data from Nephromyces and its parasitic sister taxon, Cardiosporidium, revealing an ancestral purine degradation pathway thought to have been lost early in apicomplexan evolution. The predicted localization of many of the purine degradation enzymes to peroxisomes, and the in silico identification of a full set of peroxisome proteins, indicates that loss of both features in other apicomplexans occurred multiple times. The degradation of purines is thought to play a key role in the unusual relationship between Nephromyces and its host. Transcriptome data confirm previous biochemical results of a functional pathway for the utilization of uric acid as a primary nitrogen source for this unusual apicomplexan.

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April 21, 2020

Microsatellite marker set for genetic diversity assessment of primitive Chitala chitala (Hamilton, 1822) derived through SMRT sequencing technology.

In present study, single molecule-real time sequencing technology was used to obtain a validated set of microsatellite markers for application in population genetics of the primitive fish, Chitala chitala. Assembly of circular consensus sequencing reads resulted into 1164 sequences which contained 2005 repetitive motifs. A total of 100 sequences were used for primer designing and amplification yielded a set of 28 validated polymorphic markers. These loci were used to genotype n?=?72 samples from three distant riverine populations of India, namely Son, Satluj and Brahmaputra, for determining intraspecific genetic variation. The microsatellite loci exhibited high level of polymorphism with PIC values ranging from 0.281 to 0.901. The genetic parameters revealed that mean heterozygosity ranged from 0.6802 to 0.6826 and the populations were found to be genetically diverse (Fst 0.03-0.06). This indicated the potential application of these microsatellite marker set that can used for stock characterization of C. chitala, in the wild. These newly developed loci were assayed for cross transferability in another notopterid fish, Notopterus notopterus.

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April 21, 2020

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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April 21, 2020

The red bayberry genome and genetic basis of sex determination.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high-quality genome for both a female and a male individual of red bayberry. The genome size was 313-Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole-genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59-Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59-Kb female-specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non-recombinant in the female-specific region. Sex-specific molecular markers developed from candidate genes co-segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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