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Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times., Author summary The causative agent of syphilis, Treponema pallidum subspecies pallidum, is capable of repeat infections in people, suggesting that the human immune response does not develop sufficiently broad or long-lasting immunity to cover treponemal diversity. Here, we examined the genomes from two blood-derived isolates of T. pallidum derived 6 years apart from a patient who had syphilis four times during the same period to understand the genetic basis of reinfection. We found a paucity of coding changes across the genome outside of the highly variable tprK gene. Using deep profiling of the full-length tprK gene, we found surprisingly that the two isolates did not share any full-length TprK sequences.

The repeated evolution of herbicide resistance has been cited as an example of genetic parallelism, wherein separate species or genetic lineages utilize the same genetic solution in response to selection. However, most studies that investigate the genetic basis of herbicide resistance examine the potential for changes in the protein targeted by the herbicide rather than considering genome-wide changes. We used a population genomics screen and targeted exome re-sequencing to uncover the potential genetic basis of glyphosate resistance in the common morning glory, Ipomoea purpurea, and to determine if genetic parallelism underlies the repeated evolution of resistance across replicate resistant populations. We found no evidence for changes in 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), glyphosate’s target protein, that were associated with resistance, and instead identified five genomic regions that showed evidence of selection. Within these regions, genes involved in herbicide detoxification—cytochrome P450s, ABC transporters, and glycosyltransferases—are enriched and exhibit signs of selective sweeps. One region under selection shows parallel changes across all assayed resistant populations whereas other regions exhibit signs of divergence. Thus, while it appears that the physiological mechanism of resistance in this species is likely the same among resistant populations, we find patterns of both similar and divergent selection across separate resistant populations at particular loci., Author summary Although there are many examples of herbicide resistance among natural populations of weeds, it is unknown if the same genetic mechanism underlies its repeated evolution across the landscape. Using a population genomics screen and exome re-sequencing, we examined the genetic basis of RoundUp resistance across populations of Ipomoea purpurea, a noxious agricultural weed. We identified multiple regions of the genome that exhibit signs of selection, and found genes involved in herbicide detoxification to be enriched within these regions. Interestingly, while one genomic region under selection exhibited a similar haplotype among resistant populations, other regions of the genome under selection exhibited signs of divergence. Overall, our results find evidence for both parallel and nonparallel genetic changes associated with resistance, suggesting there are more genetic avenues underlying the adaptation to herbicide than previously considered.

Background Multi-drug resistant typhoid fever remains an enormous public health threat in low and middle-income countries. However, we still lack a detailed understanding of the epidemiology and genomics of S. Typhi in many regions. Here we have undertaken a detailed genomic analysis of typhoid in urban Dhaka, Bangladesh to unravel the population structure and antimicrobial resistance patterns in S. Typhi isolated between 2004–2016. Principal findings Whole genome sequencing of 202 S. Typhi isolates obtained from three study locations in urban Dhaka revealed a diverse range of S. Typhi genotypes and AMR profiles. The bacterial population within Dhaka were relatively homogenous with little stratification between different healthcare facilities or age groups. We also observed evidence of exchange of Bangladeshi genotypes with neighboring South Asian countries (India, Pakistan and Nepal) suggesting these are circulating throughout the region. This analysis revealed a decline in H58 (genotype 4.3.1) isolates from 2011 onwards, coinciding with a rise in a diverse range of non-H58 genotypes and a simultaneous rise in isolates with reduced susceptibility to fluoroquinolones, potentially reflecting a change in treatment practices. We identified a novel S. Typhi genotype, subclade 3.3.2 (previously defined only to clade level, 3.3), which formed two localized clusters (3.3.2.Bd1 and 3.3.2.Bd2) associated with different mutations in the Quinolone Resistance Determining Region (QRDR) of gene gyrA. Significance Our analysis of S. Typhi isolates from urban Dhaka, Bangladesh isolated over a twelve year period identified a diverse range of AMR profiles and genotypes. The observed increase in non-H58 genotypes associated with reduced fluoroquinolone susceptibility may reflect a change in treatment practice in this region and highlights the importance of continued molecular surveillance to monitor the ongoing evolution of AMR in Dhaka. We have defined new genotypes and lineages of Bangladeshi S. Typhi which will facilitate the identification of these emerging AMR clones in future surveillance efforts., Author summary Typhoid fever, caused by Salmonella enterica serovar Typhi, is an acute and often life-threatening febrile illness in developing countries. Until recently, there have been limited studies focusing on the epidemiology and disease burden of typhoid in poor resource settings including Bangladesh. This study highlights the urgent need for sustained genomics based surveillance studies to monitor the population structure and ongoing evolution of AMR. Our data revealed a diverse range of S. Typhi genotypes and AMR patterns among 202 isolates collected from three urban areas in Dhaka, Bangladesh. Moreover, we defined a novel genotype, subclade 3.3.2 (previously typed only to clade level, 3.3) with two Bangladesh-localized clades 3.3.2.Bd1 and 3.3.2.Bd2 showing reduced susceptibility to fluoroquinolones. Our data shows a significant increase in non-H58 genotypes carrying QRDR mutations from 2012 onwards, replacing MDR H58 genotypes. Our data suggest that a shift in treatment practice towards third generation cephalosporins to control typhoid may be beneficial, in addition to the introduction of vaccination programs and improvements in water sanitation and hygiene (WASH) in urban Dhaka, Bangladesh.

Background:\ud Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease.\ud \ud Methods:\ud We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993–1996; n = 293] and IID2 [isolates from 2008–2009; n = 93]), the INTEGRATE project [isolates from 2016–2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163].\ud \ud Results:\ud There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation.\ud \ud Discussion:\ud Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to over 300,000 among three of the largest repositories: iGEM, Addgene, and DNASU. A challenge in biodesign remains how to use these and other repository-based sequences effectively, correctly, and seamlessly. This work describes an approach to plasmid design where a plasmid is specified as simply a DNA sequence or list of features. The proposed software then finds the most cost-effective combination of synthetic and PCR-prepared repository fragments to build the plasmid via Gibson assembly®. It finds existing DNA sequences in both user-specified and public DNA databases: iGEM, Addgene, and DNASU. Such a software application is introduced and characterized against all post-2005 iGEM composite parts and all Addgene vectors submitted in 2018 and found to reduce costs by 34% versus a purely synthetic plasmid design approach. The described software will improve current plasmid assembly workflows by shortening design times, improving build quality, and reducing costs.

In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments.

Utilising a reconstructed ancestral mitochondrial genome of a clade to design hybridisation capture baits can provide the opportunity for recovering mitochondrial sequences from all its descendent and even sister lineages. This approach is useful for taxa with no extant close relatives, as is often the case for rare or extinct species, and is a viable approach for the analysis of historical museum specimens. Asiatic linsangs (genus Prionodon) exemplify this situation, being rare Southeast Asian carnivores for which little molecular data is available. Using ancestral capture we recover partial mitochondrial genome sequences for seven banded linsangs (P. linsang) from historical specimens, representing the first intraspecific genetic dataset for this species. We additionally assemble a high quality mitogenome for the banded linsang using shotgun sequencing for time-calibrated phylogenetic analysis. This reveals a deep divergence between the two Asiatic linsang species (P. linsang, P. pardicolor), with an estimated divergence of ~12 million years (Ma). Although our sample size precludes any robust interpretation of the population structure of the banded linsang, we recover two distinct matrilines with an estimated tMRCA of ~1 Ma. Our results can be used as a basis for further investigation of the Asiatic linsangs, and further demonstrate the utility of ancestral capture for studying divergent taxa without close relatives.

Background Sinapis alba is a wild member of the Brassicaceae family reported to possess genetic resistance against major biotic and abiotic stresses of oilseed brassicas. However, the resistance nature of S. alba was not exploited generously due to the unavailability of usable genome sequences in public databases. Therefore, the present study was conducted to assemble the first draft genome from raw whole genome shotgun sequences with annotation and develop simple sequence repeat markers for molecular genetics and marker-assisted breeding. Results The raw genome sequences had 96x coverage on the Illumina platform with 170 Gbp data. The developed assembly by SOAPdenovo2 has ~459 Mbp genome size covered in 403,423 contigs with an average size of 1138.04 bp. The assembly was BLASTX with Arabidopsis thaliana which showed 32.9% positive hits between both plants. The top hit species distribution analysis showed the highest similarity with A. thaliana. A total of 809,597 GO level annotations were recorded after BLASTX results, and 34,012 sequences were annotated with different enzyme codes grouped under seven classes. The gene prediction tool AUGUSTUS identified 113,107 probable genes with an average size of 684 bp. The biochemical pathway annotation assigned 16,119 potential genes to 152 KEGG maps and 1751 enzyme codes. The development of potential SSRs from the de-novo assembly yielded 70731 unique primer pairs. Out of 159 randomly selected SSR markers for validation, 149 successfully amplified in S. alba. However, 10 SSR markers did not amplify during the validation experiment. Conclusion The annotated genome assembly with a large number of SSRs was developed in the present study. To the best of our knowledge, this is the first report of S. alba genome assembly development, annotation, and SSRs mining to date. The data presented here will be a very important resource for future crop improvement programs, especially for resistant breeding.

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain beta-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection., Author summary Determination of host specificity is one of the most significant themes in the field of infectious diseases. Identification of molecules related to the host specificity of pathogens can lead to new treatment and prevention methods, and prediction of potential host shifts. Entamoeba histolytica is a human parasite that causes hemorrhagic diarrhea, amebic colitis, and liver abscess, which may result in death in severe cases. Entamoeba nuttalli is the closest species to E. histolytica and infects various wild macaques as natural hosts. E. nuttalli might also be a pathogen with a zoonotic hazard because severe inflammatory reactions in hamster livers and an asymptomatic case of human infection have been recorded. Here, we report E. nuttalli genomic data with a quality that allows comparison with genomes from other Entamoeba species. Comparative genomics revealed common proteins shared with other mammalian Entamoeba species, as well as E. nuttalli-specific proteins. Therefore, this study identifies candidate molecules required for host specificity and subsequent pathogenicity of Entamoeba species.

Background Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates. Methods Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro. Results EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules. Conclusions EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design., Author summary Enterotoxigenic Escherichia coli (ETEC) are a significant cause of childhood diarrhea in low-middle income countries for which there is currently no vaccine. ETEC vaccine approaches to date have focused on antigens known as colonization factors (CFs) and the pathovar-defining toxins. However, both antigenic diversity of CFs and the lack of characterized CF antigens in many strains has hindered deployment of broadly protective ETEC vaccines. Nevertheless, more recent studies have identified additional ETEC virulence molecules that could compliment canonical vaccine strategies. Here, we demonstrate that two proteins, the EtpA adhesin and EatA mucinase, are present in a diverse, global collection of isolates, and are among the most highly conserved ETEC pathovar-specific antigens to be identified, providing additional incentive to explore their utility in the rational design of broadly protective ETEC vaccines.

In 2010, the rare OP354-like P[8]b rotavirus subtype was detected in children less than 2 years old in Ghana. In this follow-up study, to provide insight into the evolutionary history of the genome of Ghanaian P[8]b strains RVA/Human-wt/GHA/GHDC949/2010/G9P[8] and RVA/Human-wt/GHA/GHM0094/2010/G9P[8] detected in an infant and a 7-month old child hospitalised for acute gastroenteritis, we sequenced the complete genome using both Sanger sequencing and Illumina MiSeq technology followed by phylogenetic analysis of the near-full length sequences. Both strains possessed the Wa-like/genotype 1 constellation G9P[8]b-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence comparison and phylogenetic inference showed that both strains were identical at the lineage level throughout the 11 genome segments. Their VP7 sequences belonged to the major sub-lineage of the G9-lineage III whereas their VP4 sequences belonged to P[8]b cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9P[8]b strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either P[8]a or P[8]b mostly of African RVA origin. The introduction of a P[8]b subtype VP4 gene into the stable Wa-like strain backbone may result in strains that might propagate easily in the human population, with a potential to become an important public health concern, especially because it is not certain if the monovalent rotavirus vaccine (Rotarix) used in Ghana will be efficacious against such strains. Our analysis of the full genomes of GHM0094 and GHDC949 adds to knowledge of the genetic make-up and evolutionary dynamics of P[8]b rotavirus strains.

Amoebophrya is part of an enigmatic, diverse, and ubiquitous marine alveolate lineage known almost entirely from anonymous environmental sequencing. Two cultured Amoebophrya strains grown on core dinoflagellate hosts were used for transcriptome sequencing. BLASTx using different genetic codes suggests that Amoebophyra sp. ex Karlodinium veneficum uses the three typical stop codons (UAA, UAG, and UGA) to encode amino acids. When UAA and UAG are translated as glutamine about half of the alignments have better BLASTx scores, and when UGA is translated as tryptophan one fifth have better scores. However, the sole stop codon appears to be UGA based on conserved genes, suggesting contingent translation of UGA. Neither host sequences, nor sequences from the second strain, Amoebophrya sp. ex Akashiwo sanguinea had similar results in BLASTx searches. A genome survey of Amoebophyra sp. ex K. veneficum showed no evidence for transcript editing aside from mitochondrial transcripts. The dynein heavy chain (DHC) gene family was surveyed and of 14 transcripts only two did not use UAA, UAG, or UGA in a coding context. Overall the transcriptome displayed strong bias for A or U in third codon positions, while the tRNA genome survey showed bias against codons ending in U, particularly for amino acids with two codons ending in either C or U. Together these clues suggest contingent translation mechanisms in Amoebophyra sp. ex K. veneficum and a phylogenetically distinct instance of genetic code modification.

Spathiphyllum is a very important tropical plant used as a small, potted, ornamental plant in South China, with an annual output value of hundreds of millions of yuan. In this study, we sequenced and analyzed the complete nucleotide sequence of the Spathiphyllum 'Parrish' chloroplast genome. The whole chloroplast genome is 168,493 bp in length, and includes a pair of inverted repeat (IR) regions (IRa and IRb, each 31,600 bp), separated by a small single-copy (SSC, 15,799 bp) region and a large single-copy (LSC, 89,494 bp) region. Our annotation revealed that the S. 'Parrish' chloroplast genome contained 132 genes, including 87 protein coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. In the repeat structure analysis, we detected 281 simple sequence repeats (SSRs) which included mononucleotides (223), dinucleotides (28), trinucleotides (12), tetranucleotides (11), pentanucleotides (6), and hexanucleotides (1), in the S. 'Parrish' chloroplast genome. In addition, we identified 50 long repeats, comprising 18 forward repeats, 13 reverse repeats, 17 palindromic repeats, and 2 complementary repeats. Single nucleotide polymorphism (SNP) and insertion/deletion (indel) analyses of the chloroplast genome of the S. 'Parrish' relative S. cannifolium revealed 962 SNPs in S. 'Parrish'. There were 158 indels (90 insertions and 68 deletions) in the S. 'Parrish' chloroplast genome relative to the S. cannifolium chloroplast genome. Phylogenetic analysis of five species found S. 'Parrish' to be more closely related to S. kochii than to S. cannifolium. This study identified the characteristics of the S. 'Parrish' chloroplast genome, which will facilitate species identification and phylogenetic analysis within the genus Spathiphyllum.

A new method is introduced allowing seamless assembly of independent, functionally tested, blunt-end double strand nucleic acid parts (DNA fragments not supplied in vectors such as plasmids) into more complex biological devices (e.g. protein expression vectors) and higher order multi-device systems (e.g. biochemical pathways). Individual parts include bacterial selection markers and origins of replication, promoters useful in a variety of species, transcription terminators, shuttle sequences and a variety of "N" and "C" terminal solubility/affinity protein tags. Parts are not subjected to pre-assembly manipulation with nucleic acid modifying enzymes. Instead, they are simply mixed in appropriate pre-defined combinations and concentrations and then seamlessly linked into devices employing a specialized thermostable enzyme blend. Combinatorial assembly of parts is an inherent time-saving feature of the new method, in contrast to hierarchical binary assembly ("one part at a time") methods. This feature substantially simplifies and speeds optimization of device and system development. The versatility and functionality of the new method was shown by combinatorial assembly of parts into vector devices, one of which optimally expressed protein from a model gene. Also, a four-enzyme biosynthetic pathway system was re-created by combinatorial construction from parts and devices. Concepts discussed in this paper provide synthetic biologists, chemists and bio-engineers with improved and expanded capability to create novel biological molecules and systems.

DNA-based biodiversity analysis has gained major attention due to the use of high throughput sequencing technology in approaches such as mixed community or environmental DNA metabarcoding. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to look at how COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of site indicator taxa in benthos kick-net samples typically used in freshwater biomonitoring. We examine 6 commonly used COI primer sets, on samples collected from 6 freshwater sites. Richness is sensitive to primer choice and the combined use of additional multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. Samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Additionally, the recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Diptera may not fully capture the diversity of broadscale arthropod site indicators that can be recovered from COI metabarcoding. Based on these results, studies that use different COI amplicons may not be directly comparable. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that maximize taxon-detection or order taxa along gradients.

Criniviruses accumulate in the phloem tissue and damage crops by reducing chlorophyll which is essential for plant growth and development. Tomato chlorosis crinivirus (ToCV) is vectored by several whitefly species that damage tomato crops throughout the world. In South Africa, ToCV is a poorly studied pathogen of global economic importance. Therefore, a national survey was initiated to investigate the occurrence and distribution of criniviruses infecting tomato crops in South Africa. Whitefly infested tomato crops exhibiting interveinal leaf chlorosis and chlorotic flecking symptoms were assayed for crinivirus infections using a multiplex reverse transcription polymerase reaction (RT-PCR) approach to assess for the presence of crinivirus species that are known to infect solanaceous hosts. Next-generation sequencing (NGS) was used to generate the complete genome of ToCV from South Africa. Results from the survey indicated that ToCV is presently the only crinivirus species infecting tomatoes in South Africa. Blast analysis showed that the RNA-1 segment of ToCV from South Africa (ToCR1-186) matched 99% to Spanish isolates. On the other hand, the RNA-2 (ToCR2-186) segment matched 98% to a South Korean isolate and three Spanish isolates. Although recombination events were not detected, phylogenetic studies showed inconsistencies in the grouping of RNA-1 and RNA-2 segments for some of the ToCV isolates analyzed in this study. Therefore, we suggest the possibility of intraspecific reassortment. This is the first comprehensive study and full genome sequence of ToCV from South Africa. The information generated from this study is intended to raise awareness of ToCV infections on tomato crops in South Africa.

Chinese cabbage (Brassica rapa ssp. pekinensis) is a major crop that is widely cultivated, especially in Korea, Japan, and China. With the advent of next generation sequencing technology, the cost and time required for sequencing have decreased and the development of genome research accelerated. Genome sequencing of Chinese cabbage was completed in 2011 using the variety Chiifu-401-42, and since then the genome has been continuously updated. In the present study, we conducted whole-genome sequencing of Chinese cabbage inbred line CT001, a line widely used in traditional or molecular breeding, to improve the accuracy of genetic polymorphism analysis. The constructed CT001 pseudomolecule represented 85.4% (219.8 Mb) of the Chiifu reference genome, and a total of 38,567 gene models were annotated using RNA-Seq analysis. In addition, the spontaneous mutation rate of CT001 was estimated by resequencing DNA obtained from individual plants after sexual propagation for six generations to estimate the naturally occurring variations. The CT001 pseudomolecule constructed in this study will provide valuable resources for genomic studies on Chinese cabbage.

Recent data support that the vaginal microbiota may alter mucosal pharmacokinetics (PK) of topically delivered microbicides. Our team developed an intravaginal ring (IVR) that delivers tenofovir (TFV) (8–10 mg/day) alone or with levonorgestrel (LNG) (20 ug/day). We evaluated the effect of IVRs on the vaginal microbiota, and describe how the vaginal microbiota impacts mucosal PK of TFV. CONRAD A13-128 was a randomized, placebo controlled phase I study. We randomized 51 women to TFV, TFV/LNG or placebo IVR. We assessed the vaginal microbiota by sequencing the V3-V4 regions of 16S rRNA genes prior to IVR insertion and after approximately 15 days of use. We measured the concentration of TFV in the cervicovaginal (CV) aspirate, and TFV and TFV-diphosphate (TFV-DP) in vaginal tissue at the end of IVR use. The change in relative or absolute abundance of vaginal bacterial phylotypes was similar among active and placebo IVR users (all q values >0.13). TFV concentrations in CV aspirate and vaginal tissue, and TFV-DP concentrations in vaginal tissue were not significantly different among users with community state type (CST) 4 versus those with Lactobacillus dominated microbiota (all p values >0.07). The proportions of participants with CV aspirate concentrations of TFV >200,000 ng/mL and those with tissue TFV-DP concentrations >1,000 fmol/mg were similar among women with anaerobe versus Lactobacillus dominated microbiota (p = 0.43, 0.95 respectively). There were no significant correlations between the CV aspirate concentration of TFV and the relative abundances of Gardnerella vaginalis or Prevotella species. Tissue concentrations of TFV-DP did not correlate with any the relative abundances of any species, including Gardnerella vaginalis. In conclusion, active IVRs did not differ from the placebo IVR on the effect on the vaginal microbiota. Local TFV and TFV-DP concentrations were high and similar among IVR users with Lactobacillus dominated microbiota versus CST IV vaginal microbiota. Trial registration: ClinicalTrials.gov NCT02235662.

Mussels of the genus Bathymodiolus are among the most widespread colonizers of hydrothermal vent and cold seep environments, sustained by endosymbiosis with chemosynthetic bacteria. Presumed species of Bathymodiolus are abundant at newly discovered cold seeps on the Mid-Atlantic continental slope, however morphological taxonomy is challenging, and their phylogenetic affinities remain unestablished. Here we used mitochondrial sequence to classify species found at three seep sites (Baltimore Canyon seep (BCS; ~400m); Norfolk Canyon seep (NCS; ~1520m); and Chincoteague Island seep (CTS; ~1000m)). Mitochondrial COI (N = 162) and ND4 (N = 39) data suggest that Bathymodiolus childressi predominates at these sites, although single B. mauritanicus and B. heckerae individuals were detected. As previous work had suggested that methanotrophic and thiotrophic interactions can both occur at a site, and within an individual mussel, we investigated the symbiont communities in gill tissues of a subset of mussels from BCS and NCS. We constructed metabarcode libraries with four different primer sets spanning the 16S gene. A methanotrophic phylotype dominated all gill microbial samples from BCS, but sulfur-oxidizing Campylobacterota were represented by a notable minority of sequences from NCS. The methanotroph phylotype shared a clade with globally distributed Bathymodiolus spp. symbionts from methane seeps and hydrothermal vents. Two distinct Campylobacterota phylotypes were prevalent in NCS samples, one of which shares a clade with Campylobacterota associated with B. childressi from the Gulf of Mexico and the other with Campylobacterota associated with other deep-sea fauna. Variation in chemosynthetic symbiont communities among sites and individuals has important ecological and geochemical implications and suggests shifting reliance on methanotrophy. Continued characterization of symbionts from cold seeps will provide a greater understanding of the ecology of these unique environments as well and their geochemical footprint in elemental cycling and energy flux.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes., Author summary Our routine ability to add or alter genes in plant genomes using transgenesis has proven to be a game changer to plant sciences. Transgenics not only enables the study of gene function but also allows the development of modern crop plants without the unwanted genetic baggage coming from natural crossing. A major tool to create transgenics is the Agrobacterium system which naturally shuttles and integrates pieces of foreign DNA into its host genome. While the position and number of integrations was relatively easy to track, molecular tools never allowed to see the integrated piece of DNA within a single “picture”. Here we have utilized state-of-the-art DNA sequencing technology to capture the size and structure of multiple DNA insertion events in a plant genome. We discovered that insertion of the anticipated DNA fragment occurred as multiple concatenated full and partial fragments that led in some cases to intra- and interchromosomal rearrangements. Our analysis of the epigenetic landscapes showed variable effects from silencing of the integrated foreign DNA to alterations of chromatin marks and thus chromatin structure and functionality.

Many plants have natural partnerships with microbes that can boost their nitrogen (N) and/or phosphorus (P) acquisition. To assess whether wheat may have undiscovered associations of these types, we tested if N/P-starved Triticum aestivum show microbiome profiles that are simultaneously different from those of N/P-amended plants and those of their own bulk soils. The bacterial and fungal communities of root, rhizosphere, and bulk soil samples from the Historical Dryland Plots (Lethbridge, Canada), which hold T. aestivum that is grown both under N/P fertilization and in conditions of extreme N/P-starvation, were taxonomically described and compared (bacterial 16S rRNA genes and fungal Internal Transcribed Spacers - ITS). As the list may include novel N- and/or P-providing wheat partners, we then identified all the operational taxonomic units (OTUs) that were proportionally enriched in one or more of the nutrient starvation- and plant-specific communities. These analyses revealed: a) distinct N-starvation root and rhizosphere bacterial communities that were proportionally enriched, among others, in OTUs belonging to families Enterobacteriaceae, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Cytophagaceae, Streptomycetaceae, b) distinct N-starvation root fungal communities that were proportionally enriched in OTUs belonging to taxa Lulworthia, Sordariomycetes, Apodus, Conocybe, Ascomycota, Crocicreas, c) a distinct P-starvation rhizosphere bacterial community that was proportionally enriched in an OTU belonging to genus Agrobacterium, and d) a distinct P-starvation root fungal community that was proportionally enriched in OTUs belonging to genera Parastagonospora and Phaeosphaeriopsis. Our study might have exposed wheat-microbe connections that can form the basis of novel complementary yield-boosting tools.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

Whole genome sequencing can provide essential public health information. However, it is now known that widely used short-read methods have the potential to miss some randomly-distributed segments of genomes. This can prevent phages, plasmids, and virulence factors from being detected or properly identified. Here, we compared assemblies of three complete Shiga toxin-producing Escherichia coli (STEC) O26:H11/H- genomes from two different sequence types (ST21 and 29), each acquired using the Nextera XT MiSeq, MinION nanopore-based sequencing, and Pacific Biosciences (PacBio) sequencing. Each closed genome consisted of a single chromosome, approximately 5.7 Mb for CFSAN027343, 5.6 Mb for CFSAN027346, and 5.4 MB for CFSAN027350. However, short-read whole genome sequencing (WGS) using Nextera XT MiSeq failed to identify some virulence genes in plasmids and on the chromosome, both of which were detected using the long-read platforms. Results from long-read MinION and PacBio allowed us to identify differences in plasmid content: a single 88 kb plasmid in CFSAN027343; a 157kb plasmid in CFSAN027350; and two plasmids in CFSAN027346 (one 95 Kb, one 72 Kb). These data enabled rapid characterization of the virulome, detection of antimicrobial genes, and composition/location of Stx phages. Taken together, positive correlations between the two long-read methods for determining plasmids, virulome, antimicrobial resistance genes, and phage composition support MinION sequencing as one accurate and economical option for closing STEC genomes and identifying specific virulence markers.

Chardonnay is the basis of some of the world’s most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding., Author summary Phenotypic variation within a grapevine cultivar arises from an accumulation of mutations from serial vegetative propagation. Old cultivars such as Chardonnay have been propagated for centuries resulting in hundreds of available ‘clones’ containing unique genetic mutations and a range of various phenotypic peculiarities. The genetic mutations can be leveraged as genetic markers and are useful in identifying specific clones for authenticity testing, or as breeding markers for new clonal selections where particular mutations are known to confer a phenotypic trait. We produced a high-quality genome assembly for Chardonnay, and using re-sequencing data for 15 popular clones, were able to identify a large selection of markers that are unique to at least one clone. We identified mutations that may confer phenotypic effects, and were able to identify clones from material independently sourced from nurseries and vineyards. The marker detection framework we describe for authenticity testing would be applicable to other grapevine cultivars or even other agriculturally important woody-plant crops that are vegetatively propagated such as fruit orchards. Finally, we show that the Chardonnay genome contains extensive evidence for parental inbreeding, such that its parents, Gouais blanc and Pinot noir, may even represent first-degree relatives.

Amplification and sequencing of conserved genetic barcodes such as the cpn60 gene is a common approach to determining the taxonomic composition of microbiomes. Exact sequence variant calling has been proposed as an alternative to previously established methods for aggregation of sequence reads into operational taxonomic units (OTU). We investigated the utility of variant calling for cpn60 barcode sequences and determined the minimum sequence length required to provide species-level resolution. Sequence data from the 5´ region of the cpn60 barcode amplified from the human vaginal microbiome (n = 45), and a mock community were used to compare variant calling to de novo assembly of reads, and mapping to a reference sequence database in terms of number of OTU formed, and overall community composition. Variant calling resulted in microbiome profiles that were consistent in apparent composition to those generated with the other methods but with significant logistical advantages. Variant calling is rapid, achieves high resolution of taxa, and does not require reference sequence data. Our results further demonstrate that 150 bp from the 5´ end of the cpn60 barcode sequence is sufficient to provide species-level resolution of microbiota.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteraemia worldwide, and a model organism for the study of host-pathogen interactions. Two S. Typhimurium strains (SL1344 and ATCC14028) are widely used to study host-pathogen interactions, yet genotypic variation results in strains with diverse host range, pathogenicity and risk to food safety. The population structure of diverse strains of S. Typhimurium revealed a major phylogroup of predominantly sequence type 19 (ST19) and a minor phylogroup of ST36. The major phylogroup had a population structure with two high order clades (α and β) and multiple subclades on extended internal branches, that exhibited distinct signatures of host adaptation and anthropogenic selection. Clade α contained a number of subclades composed of strains from well characterized epidemics in domesticated animals, while clade β contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and β was reflected by the distinct distribution of antimicrobial resistance (AMR) genes, accumulation of hypothetically disrupted coding sequences (HDCS), and signatures of functional diversification. These observations were consistent with elevated anthropogenic selection of clade α lineages from adaptation to circulation in populations of domesticated livestock, and the predisposition of clade β lineages to undergo adaptation to an invasive lifestyle by a process of convergent evolution with of host adapted Salmonella serotypes. Gene flux was predominantly driven by acquisition and recombination of prophage and associated cargo genes, with only occasional loss of these elements. The acquisition of large chromosomally-encoded genetic islands was limited, but notably, a feature of two recent pandemic clones (DT104 and monophasic S. Typhimurium ST34) of clade α (SGI-1 and SGI-4)., Author summary Salmonella Typhimurium is a leading cause of foodborne illness worldwide. Our current understanding of the biology of Salmonella is largely based on studies using just two laboratory strains of S. Typhimurium, with similar characteristics. Yet this pathogen exhibits a remarkable diversity in host range, outcome of infection, and risk to human health. To investigate the genetic basis of this diversity, we have explored the genetic relationship of a collection of isolates that represent a substantial portion of the diversity of S. Typhimurium, using whole genome sequencing. S. Typhimurium evolved forming two major groups that differ in their distribution in livestock and wild avian species. The livestock-associated group contained isolates commonly affecting human health and were often drug resistant, while the wild avian-associated group was rarely associated with drug resistance and less frequently associated with human infection. We report distinct evolutionary processes acting on subgroups of S. Typhimurium including loss of information content of genomes, and gain or loss of genes predicted to affect functions such as antimicrobial resistance, disease potential, and environmental survival, and functional diversification. This study provides a framework in which to understand the epidemiology of Salmonella and improve assessment of its risk to animal and human health.

Transcriptome resources can facilitate to increase yield and quality of walnuts. Finding the best transcriptome assembly has not been the subject of walnuts research as yet. This research generated 240,179,782 reads from 11 walnut leaves according to cDNA libraries. The reads provided a complete de novo transcriptome assembly. Fifteen different transcriptome assemblies were constructed from five different well-known assemblers used in scientific literature with different k-mer lengths (Bridger, BinPacker, SOAPdenovo-Trans, Trinity and SPAdes) as well as two merging approaches (EvidentialGene and Transfuse). Based on the four quality metrics of assembly, the results indicated an efficiency in the process of merging the assemblies after being generated by de novo assemblers. Finally, EvidentialGene was recognized as the best assembler for the de novo assembly of the leaf transcriptome in walnut. Among a total number of 183,191 transcripts which were generated by EvidentialGene, there were 109,413 transcripts capable of protein potential (59.72%) and 104,926 were recognized as ORFs (57.27%). In addition, 79,185 transcripts were predicted to exist with at least one hit to the Pfam database. A number of 3,931 transcription factors were identified by BLAST searching against PlnTFDB. Furthermore, 6,591 of the predicted peptide sequences contained signaling peptides, while 92,704 contained transmembrane domains. Comparison of the assembled transcripts with transcripts of the walnut and published genome assembly for the 'Chandler' cultivar using the BLAST algorithm led to identify a total number of 27,304 and 19,178 homologue transcripts, respectively. De novo transcriptomes in walnut leaves can be developed for the future studies in functional genomics and genetic studies of walnuts.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

The low diversity of the D-subgenome of bread wheat requires the involvement of new alleles for breeding. In grasses, the allelic state of Growth Regulating Factor (GRF) gene is correlated with nitrogen uptake. In this study, we characterized the sequence of TaGRF-2D and assessed its diversity in bread wheat and goatgrass Aegilops tauschii (genome DD). In silico analysis was performed for reference sequence searching, primer pairs design and sequence assembly. The gene sequence was obtained using Illumina and Sanger sequencing. The complete sequences of TaGRF-2D were obtained for 18 varieties of wheat. The polymorphism in the presence/absence of two GCAGCC repeats in 5' UTR was revealed and the GRF-2D-SSR marker was developed. Our results showed that the alleles 5' UTR-250 and 5' UTR-238 were present in wheat varieties, 5' UTR-250 was presented in the majority of wheat varieties. In Ae. tauschii ssp. strangulata (likely donor of the D-subgenome of polyploid wheat), most accessions carried the 5' UTR-250 allele, whilst most Ae. tauschii ssp. tauschii have 5' UTR-244. The developed GRF-2D-SSR marker can be used to study the genetic diversity of wheat and Ae. tauschii.

Acinetobacter baumannii is an opportunistic pathogen of intensive care unit (ICU) patients. A. baumannii colonizes many parts of the body including the gastrointestinal tract. Endemic and epidemic strains are polyclonal. There is no clarity on the origin of polyclonality of A. baumannii. The objective of the study was to define the genetic relatedness of serial isolates and the origin of polyclonality. Serial rectal isolates from ICU patients whose rectum was colonized on ≥5 sampling occasions were selected. From a total of 32 eligible colonized patients, isolates from a subgroup of 13 patients (a total of 108 isolates) showing different patterns of colonization as revealed by pulsed-field gel electrophoresis (PFGE) were studied. The isolates were analyzed by PFGE pulsotypes, sequence types (STs) by multi-locus sequence typing (MLST) and clonal complex (CC) by eBURST analysis. Serial isolates constituted a mixture of identical, related and unrelated pulsotypes. Analysis by STs and CCs were less discriminatory. The data suggest a combination of an initial colonizing isolate undergoing mutation as well as colonization by independent isolates. Further clarity on the origin of diversity should be better obtained by whole-genome sequencing.

BackgroundMGISEQ-2000 developed by MGI Tech Co. Ltd. (a subsidiary of the BGI Group) is a new competitor of such next-generation sequencing platforms as NovaSeq and HiSeq (Illumina). Its sequencing principle is based on the DNB and cPAS technologies, which were also used in the previous version of the BGISEQ-500 device. However, the reagents for MGISEQ-2000 have been refined and the platform utilizes updated software. The cPAS method is an advanced technology based on cPAL previously created by Complete Genomics.ResultIn this paper, the authors compare the results of the whole-genome sequencing of a DNA sample from a Russian female donor performed on MGISEQ-2000 and Illumina HiSeq 2500 (both PE150). Two platforms were compared in terms of sequencing quality, number of errors and performance. Additionally, we performed variant calling using four different software packages: Samtools mpileaup, Strelka2, Sentieon, and GATK. The accuracy of single nucleotide polymorphism (SNP) detection was similar in the data generated by MGISEQ-2000 and HiSeq 2500, which was used as a reference. At the same time, a separate indel analysis of the overall error rate revealed similar FPR values and lower sensitivity.Conclusionsit may be concluded with confidence that the data generated by the analyzed sequencing systems is characterized by comparable magnitudes of error and that MGISEQ-2000 can be used for a wide range of research tasks on par with HiSeq 2500.

The Amphiprion (anemonefish or clownfish) family of teleost fish, which is not a common model species, exhibits multiple unique characteristics, including social control of body size and protandrous sex change. The social changes in sex and body size are modulated by neuropeptide signaling pathways. These neuropeptides are formed from complex processing from larger prohormone proteins; understanding the neuropeptide complement requires information on complete prohormones sequences. Genome and transcriptome information within and across 22 teleost fish species, including 11 Amphiprion species, were assembled and integrated to achieve the first comprehensive survey of their prohormone genes. This information enabled the identification of 175 prohormone isoforms from 159 prohormone proteins across all species. This included identification of 9 CART prepropeptide genes and the loss of insulin-like 5B and tachykinin precursor 1B genes in Pomacentridae species. Transcriptome assemblies generally detected most prohormone genes but provided fewer prohormone genes than genome assemblies due to the lack of expression of prohormone genes or specific isoforms and tissue sampled. Comparisons between duplicate genes indicated that subfunctionalization, degradation, and neofunctionalization may be occurring between all copies. Characterization of the prohormone complement lays the foundation for future peptidomic investigation of the molecular basis of social physiology and behavior in the teleost fish.

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study.

Detailed comprehensive knowledge of the structures of individual long-range telomere-terminal haplotypes are needed to understand their impact on telomere function, and to delineate the population structure and evolution of subtelomere regions. However, the abundance of large evolutionarily recent segmental duplications and high levels of large structural variations have complicated both the mapping and sequence characterization of human subtelomere regions. Here, we use high throughput optical mapping of large single DNA molecules in nanochannel arrays for 154 human genomes from 26 populations to present a comprehensive look at human subtelomere structure and variation. The results catalog many novel long-range subtelomere haplotypes and determine the frequencies and contexts of specific subtelomeric duplicons on each chromosome arm, helping to clarify the currently ambiguous nature of many specific subtelomere structures as represented in the current reference sequence (HG38). The organization and content of some duplicons in subtelomeres appear to show both chromosome arm and population-specific trends. Based upon these trends we estimate a timeline for the spread of these duplication blocks., Author summary The ends of human chromosomes have caps called telomeres that are essential. These telomeres are influenced by the portions of DNA next to them, a region known as the subtelomere. We need to better understand the subtelomeric region to understand how it impacts the telomeres. This subtelomeric region is not well described in the current references. This is due to large variations in this region and portions that are repeated many times, making current sequencing technologies struggle to capture these regions. Many of these variations are evolutionary recent. Here we use 154 different samples from the 26 geographic regions of the world to gain a better understanding of the variation in these regions. We found many new haplotypes and clarified the haplotypes existing in the current reference. We then examined population and chromosome specific trends.

The discovery of hepaciviruses in non-human hosts has accelerated following the advancement of high-throughput sequencing technology. Hepaciviruses have now been described in reptiles, fish, birds, and an extensive array of mammals. Using metagenomic sequencing on pooled samples of field-collected Culex annulirostris mosquitoes, we discovered a divergent hepacivirus-like sequence, named Jogalong virus, from the Kimberley region in northern Western Australia. Using PCR, we screened the same 300 individual mosquitoes and found just a single positive sample (1/300, 0.33%). Phylogenetic analysis of the hepacivirus NS5B protein places Jogalong virus within the genus Hepacivirus but on a distinct and deeply rooted monophyletic branch shared with duck hepacivirus, suggesting a notably different evolutionary history. Vertebrate barcoding PCR targeting two mitochondrial genes, cytochrome c oxidase subunit I and cytochrome b, indicated that the Jogalong virus-positive mosquito had recently fed on the tawny frogmouth (Podargus strigoides), although it is currently unknown whether this bird species contributes to the natural ecology of this virus.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism., Author summary How a genome folds within a nucleus can contribute to the regulation of gene expression. There is now considerable interest in understanding how the three-dimensional structure of a genome is maintained and how this structure influences biological processes. However, most studies on these phenomena focus on a few model organisms, which limits our knowledge of how the nuclear organisation is maintained and the range of biological processes to which it contributes. In this paper, we examine the three-dimensional organisation of the genome in Epichloë festucae, a symbiotic fungus. Our results demonstrate that repetitive elements play a key role in shaping the nuclear organisation of E. festucae. These repetitive elements help to divide the genome into distinct regions that have similar gene expression profiles. Our results demonstrate a novel role for repeats in maintaining a three-dimensional organisation that contributes to regulating the symbiotic relationship formed by this species.

Mitochondrial disorders arise from defects in nuclear genes encoding enzymes of oxidative metabolism. Mutations of metabolic enzymes in somatic tissues can cause cancers due to oncometabolite accumulation. Paraganglioma and pheochromocytoma are examples, whose etiology and therapy are complicated by the absence of representative cell lines or animal models. These tumors can be driven by loss of the tricarboxylic acid cycle enzyme succinate dehydrogenase. We exploit the relationship between succinate accumulation, hypoxic signaling, egg-laying behavior, and morphology in C. elegans to create genetic and pharmacological models of succinate dehydrogenase loss disorders. With optimization, these models may enable future high-throughput screening efforts.

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified inSalmonellaTyphi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758Salmonellapublic genomes. Pangenome analysis of pBSSB1-family contigs was performed using Roary and we identified three core genes amenable to a minimal MLST scheme. Population structure analysis based on the newly developed MLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This MLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name “pBSSB1-family”.

DNA metabarcoding assays are powerful tools for delving into the DNA in wildlife feces, giving unprecedented ability to detect species, understand natural history, and identify pathogens for a range of applications in management, conservation, and research. Next-generation sequencing technology is developing rapidly, which makes it especially important that predictability and reproducibility of DNA metabarcoding assays are explored together with the post-depositional ecology of the target taxon’s fecal DNA. Here, we defined the constraints of an assay called ‘Species from Feces’ used by government agencies, research groups, and non-governmental organizations to identify bat species from guano. We tested assay sensitivity by examining how time and humidity affect the ability to recover and successfully sequence DNA in guano, assessing whether a fecal pellet from a rare bat species could be detected in a background of feces from other bat species, and evaluating the efficacy of Species from Feces as a survey tool for bat roosts in temperate and tropical areas. We found that the assay performs well with feces over two years old in dry, cool environments, and fails by 12 months at 100% relative humidity. We also found that it reliably identifies rare DNA, has great utility for surveying roosts in temperate and tropical regions, and detects more bat species than do visual surveys. We attribute the success of Species from Feces to characteristics of the assay paired with application in taxa that are particularly well-suited for fecal DNA survival. In a time of rapid evolution of DNA metabarcoding approaches and their use with feces, this study illustrates the strengths and limitations of applied assays.

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP’s prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles., Author summary With the advent and evolution of next-generation sequencing technology, efforts to identify and catalog the expressed RNA molecules have greatly advanced our understanding of RNA biology. However, the current standard RNA-seq methods, particularly those targeting short ncRNAs, do not fully capture all of the RNAs expressed but allow for some “escapers” to slip. RNAs generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends, and those cP-containing RNAs (cP-RNAs) are one such escaper that are not ligated to a 3′-adapter and thus uncaptured by standard RNA-seq. Although an increasing number of studies has been suggesting their functional significances, cP-RNAs remained a hidden component in the transcriptome, infrequently recognized and characterized. In this study, we provide the first genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. By using cP-RNA-seq technique that can specifically sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from tRNAs, mRNAs, and rRNAs. cP-RNAs are generated by previously-uncharacterized, highly-specific RNA cleavage events between cytidine and adenosine, which is regulated through aging. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer., Author summary Bdelloid rotifers are microscopic animals that live in freshwater habitats throughout the world. Two life history characteristics distinguish these common invertebrates as extraordinary. First, they have existed for millions of years apparently without sex: males have not been reported, and females produce genetically identical daughters via parthenogenesis. Second, most bdelloid species are highly resilient to desiccation and can survive without water for extended periods of time (a process known as anhydrobiosis). These 2 attributes have been predicted to leave signatures in bdelloid genomes. Here, we present new draft genomes for 3 bdelloid species and employ comparative genomics to explore the potential impacts of bdelloids’ unusual lifestyle on genome structure and content. We find that many proposed genomic consequences of asexuality and desiccation tolerance do not hold true for all species. The genomes of bdelloids may be more similar to those of other animals than previously thought, though a remarkable exception is the high proportion of genes acquired horizontally from nonmetazoan taxa. Our findings necessitate a reevaluation of the effects of asexuality and desiccation on genome evolution in bdelloid rotifers.

Humans are a diploid species that inherit one set of chromosomes paternally and one homologous set of chromosomes maternally. Unfortunately, most human sequencing initiatives ignore this fact in that they do not directly delineate the nucleotide content of the maternal and paternal copies of the 23 chromosomes individuals possess (i.e., they do not ‘phase’ the genome) often because of the costs and complexities of doing so. We compared 11 different widely-used approaches to phasing human genomes using the publicly available ‘Genome-In-A-Bottle’ (GIAB) phased version of the NA12878 genome as a gold standard. The phasing strategies we compared included laboratory-based assays that prepare DNA in unique ways to facilitate phasing as well as purely computational approaches that seek to reconstruct phase information from general sequencing reads and constructs or population-level haplotype frequency information obtained through a reference panel of haplotypes. To assess the performance of the 11 approaches, we used metrics that included, among others, switch error rates, haplotype block lengths, the proportion of fully phase-resolved genes, phasing accuracy and yield between pairs of SNVs. Our comparisons suggest that a hybrid or combined approach that leverages: 1. population-based phasing using the SHAPEIT software suite, 2. either genome-wide sequencing read data or parental genotypes, and 3. a large reference panel of variant and haplotype frequencies, provides a fast and efficient way to produce highly accurate phase-resolved individual human genomes. We found that for population-based approaches, phasing performance is enhanced with the addition of genome-wide read data; e.g., whole genome shotgun and/or RNA sequencing reads. Further, we found that the inclusion of parental genotype data within a population-based phasing strategy can provide as much as a ten-fold reduction in phasing errors. We also considered a majority voting scheme for the construction of a consensus haplotype combining multiple predictions for enhanced performance and site coverage. Finally, we also identified DNA sequence signatures associated with the genomic regions harboring phasing switch errors, which included regions of low polymorphism or SNV density., Author summary Humans are a diploid species that inherit one set of chromosomes paternally and one set of chromosomes maternally. Separating the nucleotide content of the maternally and paternally-derived chromosomes for an individual, i.e., ‘phasing’ that individual’s genome, is not trivial with today’s sequencing technologies. This is in part due to the fact that most available sequencing technologies generate short sequencing reads that make it hard to assemble individual homologous chromosome pairs. Phase information can be crucial for putting into context the likely functional consequences of DNA sequence variants as well as certain evolutionary and population genetics phenomena. In order to assess the reliability of current sequencing-based phasing strategies, we compared 11 different approaches using a public domain reference genome as a test case. These phasing strategies included laboratory-based experimental techniques as well as purely computational approaches. Importantly, our comparisons show that a hybrid or combined approach that leverages population-based phasing via the SHAPEIT software suite works well and can be improved with the addition of genome-wide sequence read or parental genotype data.

Background The yellow sisorid catfish (Bagarius yarrelli) is a carnivorous freshwater fish that inhabits the Honghe River, Lanchangjiang River and Nujiang River of southern China and other Southeast Asian countries. However, the publicly available genomic data for B. yarrelli are limited. Methodology and principal findings Illumina Solexa paired-end technology produced 1,706,456 raw reads from muscle, liver and caudal fin tissues of B. yarrelli. Nearly 5 Gb of data were acquired, and de novo assembly generated 14,607 unigenes, with an N50 of 2006 bp. A total of 9093 unigenes showed significant similarities to known proteins in public databases: 4477 and 6391 of B. yarrelli unigenes were mapped to the Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases, respectively. Moreover, 9635 unigenes were assigned to 242 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, 8568 microsatellites (simple sequence repeats, SSRs) were detected, and 31 pairs of polymorphic primers were characterized using wild populations of B. yarrelli from the Nujiang River, Yunnan Province, China. Conclusion/Significance These sequences enrich the genomic resources for B. yarrelli and will benefit future investigations into the evolutionary and biological processes of this and related Bagarius species. The SSR markers developed in this study will facilitate construction of genetic maps, investigations of genetic structures and germplasm polymorphism assessments in B. yarrelli.

Bioinformatics applications and pipelines increasingly use k-mer indexes to search for similar sequences. The major problem with k-mer indexes is that they require lots of memory. Sampling is often used to reduce index size and query time. Most applications use one of two major types of sampling: fixed sampling and minimizer sampling. It is well known that fixed sampling will produce a smaller index, typically by roughly a factor of two, whereas it is generally assumed that minimizer sampling will produce faster query times since query k-mers can also be sampled. However, no direct comparison of fixed and minimizer sampling has been performed to verify these assumptions. We systematically compare fixed and minimizer sampling using the human genome as our database. We use the resulting k-mer indexes for fixed sampling and minimizer sampling to find all maximal exact matches between our database, the human genome, and three separate query sets, the mouse genome, the chimp genome, and an NGS data set. We reach the following conclusions. First, using larger k-mers reduces query time for both fixed sampling and minimizer sampling at a cost of requiring more space. If we use the same k-mer size for both methods, fixed sampling requires typically half as much space whereas minimizer sampling processes queries only slightly faster. If we are allowed to use any k-mer size for each method, then we can choose a k-mer size such that fixed sampling both uses less space and processes queries faster than minimizer sampling. The reason is that although minimizer sampling is able to sample query k-mers, the number of shared k-mer occurrences that must be processed is much larger for minimizer sampling than fixed sampling. In conclusion, we argue that for any application where each shared k-mer occurrence must be processed, fixed sampling is the right sampling method.

Xiaguan Tuo Tea is largely consumed by the Chinese, but there is little research into the microbial diversity and component changes during the fermentation of this tea. In this study, we first used fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) and chemical analysis methods to determine the microbial abundance and diversity and the chemical composition during fermentation. The FISH results showed that the total number of microorganisms ranges from 2.3×102 to 4.0×108 cells per gram of sample during fermentation and is mainly dominated by fungi. In the early fermentation stages, molds are dominant (0.6×102~2.8×106 cells/g, 0~35 d). However, in the late stages of fermentation, yeasts are dominant (3.6×104~9.6×106 cells/g, 35~56 d). The bacteria have little effect during the fermentation of tea (102~103 cells/g

In plant breeding the use of molecular markers has resulted in tremendous improvement of the speed with which new crop varieties are introduced into the market. Single Nucleotide Polymorphism (SNP) genotyping is routinely used for association studies, Linkage Disequilibrium (LD) and Quantitative Trait Locus (QTL) mapping studies, marker-assisted backcrosses and validation of large numbers of novel SNPs. Here we present the KeyGene SNPSelect technology, a scalable and flexible multiplexed, targeted sequence-based, genotyping solution. The multiplex composition of SNPSelect assays can be easily changed between experiments by adding or removing loci, demonstrating their content flexibility. To demonstrate this versatility, we first designed a 1,056-plex maize assay and genotyped a total of 374 samples originating from an F2 and a Recombinant Inbred Line (RIL) population and a maize germplasm collection. Next, subsets of the most informative SNP loci were assembled in 384-plex and 768-plex assays for further genotyping. Indeed, selection of the most informative SNPs allows cost-efficient yet highly informative genotyping in a custom-made fashion, with average call rates between 88.1% (1,056-plex assay) and 99.4% (384-plex assay), and average reproducibility rates between duplicate samples ranging from 98.2% (1056-plex assay) to 99.9% (384-plex assay). The SNPSelect workflow can be completed from a DNA sample to a genotype dataset in less than three days. We propose SNPSelect as an attractive and competitive genotyping solution to meet the targeted genotyping needs in fields such as plant breeding.

The NJAY (New Jersey aster yellows) strain of ‘Candidatus Phytoplasma asteris’ is a significant plant pathogen responsible for causing severe lettuce yellows in the U.S. state of New Jersey. A draft genome sequence was prepared for this organism. A total of 177,847 reads were assembled into 75 contigs > 518 bp with a total base value of 652,092 and an overall [G+C] content of 27.1%. A total of 733 protein coding genes were identified. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MAPF00000000. This draft genome was used for genome- and gene-based comparative phylogenetic analyses with other phytoplasmas, including the closely related ‘Ca. Phytoplasma asteris’ strain, aster yellows witches’- broom (AY-WB). NJAY and AY-WB exhibit approximately 0.5% dissimilarity at the nucleotide level among their shared genomic segments. Evidence indicated that NJAY harbors four plasmids homologous to those known to encode pathogenicity determinants in AY-WB, as well as a chromosome-encoded mobile unit. Apparent NJAY orthologs to the important AY-WB virulence factors, SAP11 and SAP54, were identified. A number of secreted proteins, both membrane-bound and soluble, were encoded, with many bearing similarity to known AY-WB effector molecules and others representing possible secreted proteins that may be novel to the NJAY lineage.

Human pathogens can survive and grow in hot springs. For water quality assessment, Escherichia coli or Enterococci are the main thermotolerant enteric bacteria commonly used to estimate the load of pathogenic bacteria in water. However, most of the environmental bacteria are unculturable thus culture methods may cause bias in detection of most pathogens. Illumina sequencing can provide a more comprehensive and accurate insight into environmental bacterial pathogens, which can be used to develop better risk assessment methods and promote public health awareness. In this study, high-throughput Illumina sequencing was used to identify bacterial pathogens from five hot springs; Maiwooi, Akwar, Garbanabra, Elegedi and Gelti, in Eritrea. Water samples were collected from the five hot springs. Total community DNA was extracted from samples using the phenol-chloroform method. The 16S rRNA gene variable region (V4—V7) of the extracted DNA was amplified and library construction done according to Illumina sequencing protocol. The sequence reads (length >200 bp) from Illumina sequencing libraries ranged from 22,091 sequences in the wet sediment sample from Garbanabra to 155,789 sequences in the mat sample from Elegedi. Taxonomy was assigned to each OTU using BLASTn against a curated database derived from GreenGenes, RDPII, SILVA SSU Reference 119 and NCBI. The proportion of potential pathogens from the water samples was highest in Maiwooi (17.8%), followed by Gelti (16.7%), Akwar (13.6%) and Garbanabra (10.9%). Although the numbers of DNA sequence reads from Illumina sequencing were very high for the Elegedi (104,328), corresponding proportion of potential pathogens very low (3.6%). Most of the potential pathogenic bacterial sequences identified were from Proteobacteria and Firmicutes. Legionella and Clostridium were the most common detected genera with different species. Most of the potential pathogens were detected from the water samples. However, sequences belonging to Clostridium were observed more abundantly from the mat samples. This study employed high-throughput sequencing technologies to determine the presence of pathogenic bacteria in the five hot springs in Eritrea.

Background Hepatocellular adenomas (HCA) are benign liver tumors that may transform into hepatocellular carcinoma (HCC), but the molecular drivers of this transformation remain ill-defined. This study evaluates the molecular changes in HCA and HCC and in comparison to their adjacent non-neoplastic liver. Methods 11 patients with HCA and 10 patients with HCC without underlying hepatitis or cirrhosis were included in this pilot study. Tumor and non-tumor liver tissues were selected for immunohistochemical staining, small RNA sequencing, and targeted gene sequencing. We compared microRNA expressions and mutations between HCA and HCC and non-neoplastic liver. Results HCA were classified as inflammatory (n = 6), steatotic (n = 4), or β-catenin activated (n = 1) subtypes. MicroRNA profile of all 3 HCA subtypes clustered between that of normal liver and HCC in principal component analysis. In both HCA and HCC, miR-200a, miR-429, and miR-490-3p were significantly downregulated compared to normal liver, whereas miR-452, miR-766, and miR-1180 were significantly upregulated. In addition, compared to HCA, HCC had significantly higher expression of members of the chromosome 19 miRNA cluster (C19MC), including miR-515-5p, miR-517a, miR-518b, and miR-520c-3p. Conclusions This study indicates that while there are significant differences in the molecular profile between HCA and HCC, several miRNAs are similarly deregulated in HCA and HCC compared to adjacent normal liver. These results may provide insights into the drivers of hepatocarcinogenesis and warrant further investigations.