Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Background Epstein–Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. Methods In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other’s data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. Results This study detected novel genes embedded into longer host genes containing 5′-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. Conclusions An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map

Background The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. Results The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92–94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. Conclusions The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Complete Genome Sequence of a Halophilic Bacterium, Halomonas sp. Strain NyZ770, from Mariana Trench Sediment

Halomonas sp. strain NyZ770 is a bacterium that was isolated from Mariana Trench sediment. Here, the complete genome sequence of this strain is reported. The genome was sequenced with the Illumina NovaSeq and Pacific Biosciences Sequel sequencing platforms and consists of a single chromosome of 4,024,853 bp, with a G+C content of 60.21%.

Genome of a Giant (Trevally): Caranx ignobilis

ABSTRACTCaranx ignobilis, commonly known as the kingfish or giant trevally, is a large, reef-associated apex predator. It is a prized sportfish, targeted heavily throughout its tropical and subtropical range in the Indian and Pacific Oceans, and it has drawn significant interest in aquaculture due to an unusual tolerance for freshwater. In this study, we present a high-quality nuclear genome assembly of a C. ignobilis individual from Hawaiian waters, which have recently been shown to host a genetically distinct population. The assembly has a contig NG50 of 7.3Mbp and scaffold NG50 of 46.3Mbp. Twenty-five of the 203 scaffolds contain 90% of the genome. We also present the raw Pacific Biosciences continuous long-reads from which the assembly was created. A Hi-C dataset (Dovetail Genomics Omni-C) and Illumina-based RNA-seq from eight tissues are also presented; the latter of which can be particularly useful for annotation and studies of freshwater tolerance. Overall, this genome assembly and supporting data is a valuable tool for ecological and comparative genomics studies of kingfish and other carangoid fishes.

A chromosome-level genome assembly for the Pacific oyster Crassostrea gigas

Background The Pacific oyster (Crassostrea gigas) is a bivalve mollusc with vital roles in coastal ecosystems and aquaculture globally. While extensive genomic tools are available for C. gigas, highly contiguous reference genomes are required to support both fundamental and applied research. Herein we report the creation and annotation of a chromosome-level assembly for C. gigas. Findings High-coverage long- and short-read sequence data generated on Pacific Biosciences and Illumina platforms were used to generate an initial assembly, which was then scaffolded into 10 pseudo-chromosomes using both Hi-C sequencing and a high-density linkage map. The assembly has a scaffold N50 of 58.4 Mb and a contig N50 of 1.8 Mb, representing a step advance on the previously published C. gigas assembly. Annotation based on Pacific Biosciences Iso-Seq and Illumina RNA-Seq resulted in identification of ∼30,000 putative protein-coding genes. Annotation of putative repeat elements highlighted an enrichment of Helitron rolling-circle transposable elements, suggesting their potential role in shaping the evolution of the C. gigas genome. Conclusions This new chromosome-level assembly will be an enabling resource for genetics and genomics studies to support fundamental insight into bivalve biology, as well as for selective breeding of C. gigas in aquaculture.

Comparison of long-read methods for sequencing and assembly of a plant genome

Background Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

Chromosome-level draft genome of a diploid plum (Prunus salicina)

Background Plums are one of the most economically important Rosaceae fruit crops and comprise dozens of species distributed across the world. Until now, only limited genomic information has been available for the genetic studies and breeding programs of plums. Prunus salicina, an important diploid plum species, plays a predominant role in modern commercial plum production. Here we selected P. salicina for whole-genome sequencing and present a chromosome-level genome assembly through the combination of Pacific Biosciences sequencing, Illumina sequencing, and Hi-C technology. Findings The assembly had a total size of 284.2 Mb, with contig N50 of 1.78 Mb and scaffold N50 of 32.32 Mb. A total of 96.56% of the assembled sequences were anchored onto 8 pseudochromosomes, and 24,448 protein-coding genes were identified. Phylogenetic analysis showed that P. salicina had a close relationship with Prunus mume and Prunus armeniaca, with P. salicina diverging from their common ancestor ∼9.05 million years ago. During P. salicina evolution 146 gene families were expanded, and some cell wall–related GO terms were significantly enriched. It was noteworthy that members of the DUF579 family, a new class involved in xylan biosynthesis, were significantly expanded in P. salicina, which provided new insight into the xylan metabolism in plums. Conclusions We constructed the first high-quality chromosome-level plum genome using Pacific Biosciences, Illumina, and Hi-C technologies. This work provides a valuable resource for facilitating plum breeding programs and studying the genetic diversity mechanisms of plums and Prunus species.

Comparison of the two up-to-date sequencing technologies for genome assembly: HiFi reads of Pacific Biosciences Sequel II system and ultralong reads of Oxford Nanopore

Background The availability of reference genomes has revolutionized the study of biology. Multiple competing technologies have been developed to improve the quality and robustness of genome assemblies during the past decade. The 2 widely used long-read sequencing providers—Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)—have recently updated their platforms: PacBio enables high-throughput HiFi reads with base-level resolution of >99%, and ONT generated reads as long as 2 Mb. We applied the 2 up-to-date platforms to a single rice individual and then compared the 2 assemblies to investigate the advantages and limitations of each. Results The results showed that ONT ultralong reads delivered higher contiguity, producing a total of 18 contigs of which 10 were assembled into a single chromosome compared to 394 contigs and 3 chromosome-level contigs for the PacBio assembly. The ONT ultralong reads also prevented assembly errors caused by long repetitive regions, for which we observed a total of 44 genes of false redundancies and 10 genes of false losses in the PacBio assembly, leading to over- or underestimation of the gene families in those long repetitive regions. We also noted that the PacBio HiFi reads generated assemblies with considerably fewer errors at the level of single nucleotides and small insertions and deletions than those of the ONT assembly, which generated an average 1.06 errors per kb and finally engendered 1,475 incorrect gene annotations via altered or truncated protein predictions. Conclusions It shows that both PacBio HiFi reads and ONT ultralong reads had their own merits. Further genome reference constructions could leverage both techniques to lessen the impact of assembly errors and subsequent annotation mistakes rooted in each.

Pacific Biosciences Single-molecule Real-time (SMRT) Sequencing Reveals High Diversity of Basal Fungal Lineages and Stochastic Processes Controlled Fungal Community Assembly in Mangrove Sediments

Background: Mangrove wetlands are unique ecosystems with specific environmental characteristics, and are a hotspot of biodiversity. Although they probably harbor a variety of mangrove-specific fungi, the compositions of mangrove fungal community has been rarely investigated in detail, except for few published culture-based studies. In addition, the fungal community assembly and interaction patterns that impact the community composition in mangroves have not been explored to date. Results: We used the Pacific Biosciences single-molecule real-time sequencing approach targeting the entire internal transcribed spacer region, to systematically investigate the composition, biogeographical patterns, assembly processes, co-occurrence patterns and shaping factors of the fungal communities in sediments of seven representative mangroves across the Southeast China. We recovered 15 phyla, including some early diverging fungal lineages not previously reported in mangroves. Phylogenetic analysis revealed an incredibly high proportion of Rozellomycota and Chytridiomycota, as accounting for up to one-third of all fungal abundance. Although the neutral community model described a moderate portion of community variation, the similarity of fungal communities exhibited strong distance-decay patterns. Furthermore, the mean values and most beta nearest-taxon index fell between -2 to 2, with Bray–Curtis-based Raup–Crick value generally greater than 0.95, suggesting that stochastic processes strongly shape the fungal community composition. Consistently, nonmetric multidimensional scaling and permutational multivariate analysis of variance confirmed the geographical location as a crucial factor driving the distribution of both, the dominant and rare taxa of mangrove fungi. The db-RDA analyses indicated a minor role of environmental selections in shaping the fungal community. Network analyses revealed that the deep sediments harbor more complex fungal networks with highly connected taxa than surface sediments, and that rare fungal taxa might play important roles in microbial interactions and ecological functions in mangrove sediments.Conclusions: The investigation revealed high fungal diversity in mangrove sediments, with incredibly high numbers of basal fungal lineages, stochastic processes driving the assembly of fungal community, and geographic location strongly shaping fungal community composition in mangroves. These discoveries therefore spur further studies of the utilization and protection of fungal resources and communities in mangrove sediments.