Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and ...phylogenetic information for species discrimination and taxonomic assignment.
Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples.
Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer–template mismatches bias the taxonomic profile when using regular and highly degenerate primers.
The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.
Genomics in the long-read sequencing era van Dijk, Erwin L.; Naquin, Delphine; Gorrichon, Kévin ...
Trends in genetics,
09/2023, Letnik:
39, Številka:
9
Journal Article
Recenzirano
Long-read sequencing (LRS) methods can now produce highly accurate (ultra)long reads and thus enable for the first time the production of truly complete telomere-to-telomere (T2T) assemblies of ...complex genomes.While the recently produced T2T assemblies were labor-intensive and required a combination of various techniques, in the near future T2T assemblies based on LRS alone will be produced.We have entered the era of population scale long-read sequencing, where graph-based pangenomes much better representing genomic variation will increasingly be used in the near future.New applications of LRS appear at a rapid pace, with examples ranging from higher order chromatin interaction studies to the quality control of mRNA vaccines.
Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.
Dental plaque is the main cause leading to the dental caries and periodontal diseases. The main purpose of this study was to test the efficacy of oral spray containing the antimicrobial peptide P-113 ...on the reduction of oral bacteria number and dental plaque formation in a randomized clinical assessment.
This study was divided into two parts. In Part A, we investigated the user experiences with the P-113 containing oral spray. In part B, 14 subjects in the experimental group used the P-113-containing oral spray, while 14 subjects in the control group used a placebo without the P-113 in a 4-week clinical trial. Participants were asked to use the P-113-containing oral spray or placebo 3 times per day and 5 times per use. Moreover, 3 check-ups and 2 washouts were carried out to evaluate the DMFT score, dental plaque weight, dental plaque index, and gingival index.
In part A, up to 91.8% of the subjects in the experimental group were satisfied with the use of the P-113-containing oral spray. In part B, based on our PacBio SMRT sequencing platform and DADA2 analysis, the numbers of Streptococcus and Porphyromonas in the experimental group were lower than those in the control group. In addition, decreased dental plaque weight, dental plaque index, and gingival index were all observed in the experimental group.
The P-113-containing oral spray has the potential to reduce the dental caries and periodontal disease-related bacteria and to control the dental plaque formation.
•In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the zebra finch brain ...transcriptome.•To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines.•We added 220 novel genes and 8134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation.•Using mass-spectrometry of zebra finch caudal telencephalon, we validated 18 different novel proteins.•Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.
The zebra finch (Taeniopygia guttata), a representative oscine songbird species, has been widely studied to investigate behavioral neuroscience, most notably the neurobiological basis of vocal learning, a rare trait shared in only a few animal groups including humans. In 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from the Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. We further validated 18 different novel proteins by using mass-spectrometry data generated from zebra finch caudal telencephalon tissue. Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.
Summary
Alternative splicing (AS) is a key post‐transcriptional regulatory mechanism, yet little information is known about its roles in fruit crops. Here, AS was globally analyzed in the wild ...strawberry Fragaria vesca genome with RNA‐seq data derived from different stages of fruit development. The AS landscape was characterized and compared between the single‐molecule, real‐time (SMRT) and Illumina RNA‐seq platform. While SMRT has a lower sequencing depth, it identifies more genes undergoing AS (57.67% of detected multiexon genes) when it is compared with Illumina (33.48%), illustrating the efficacy of SMRT in AS identification. We investigated different modes of AS in the context of fruit development; the percentage of intron retention (IR) is markedly reduced whereas that of alternative acceptor sites (AA) is significantly increased post‐fertilization when compared with pre‐fertilization. When all the identified transcripts were combined, a total of 66.43% detected multiexon genes in strawberry undergo AS, some of which lead to a gain or loss of conserved domains in the gene products. The work demonstrates that SMRT sequencing is highly powerful in AS discovery and provides a rich data resource for later functional studies of different isoforms. Further, shifting AS modes may contribute to rapid changes of gene expression during fruit set.
Significance Statement
Alternative splicing is a key post‐transcriptional regulatory mechanism, yet little is known about its roles in fruit crops. Here we globally analyzed alternative splicing using transcriptome data from strawberry fruits, and show that single molecule real time (SMRT) RNA‐seq data is highly efficient in uncovering alternatively spliced variants.
Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete ...genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, ΦAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.
•SMRT and SGS sequencing could providing a comprehensive transcriptome data for Ginkgo.•LncRNA, AS, and fusion gene participating in the regulation of flavonoid metabolism.•Revealing the regulation ...network associated with flavonoid biosynthesis in G. biloba.•Revealing the synthetic transport of flavonoids in G. biloba.
Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.
In this study, we analyzed the fermentation quality, microbial community, and metabolome characteristics of ryegrass silage from different harvests (first harvest-AK, second harvest-BK, and third ...harvest-CK) and analyzed the correlation between fermentative bacteria and metabolites. The bacterial community and metabolomic characteristics were analyzed by single-molecule real-time (SMRT) sequencing and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), respectively. After 60 days of ensiling, the pH of BK was significantly lower than those of AK and CK, and its lactic acid content was significantly higher than those of AK and CK.
Lactiplantibacillus
and
Enterococcus
genera dominate the microbiota of silage obtained from ryegrass harvested at three different harvests. In addition, the BK group had the highest abundance of
Lactiplantibacillus plantarum
(58.66%), and the CK group had the highest abundance of
Enterococcus faecalis
(42.88%). The most annotated metabolites among the differential metabolites of different harvests were peptides, and eight amino acids were dominant in the composition of the identified peptides. In the ryegrass silage, arginine, alanine, aspartate, and glutamate biosynthesis had the highest enrichment ratio in the metabolic pathway of KEGG pathway enrichment analysis. Valyl-isoleucine and glutamylvaline were positively correlated with
Lactiplantibacillus plantarum
. D-Pipecolic acid and L-glutamic acid were positively correlated with
Levilactobacillus brevis
. L-phenylalanyl-L-proline, 3,4,5-trihydroxy-6-(2-methoxybenzoyloxy) oxane-2-carboxylic acid, and shikimic acid were negatively correlated with
Levilactobacillus brevis
. In conclusion, this study explains the effects of different harvest frequencies on the fermentation quality, microbial community, and metabolites of ryegrass, and improves our understanding of the ensiling mechanisms associated with different ryegrass harvesting frequencies.
Microorganisms are ubiquitous, and those inhabiting plants have been the subject of several studies. Plant-associated bacteria exhibit various biological mechanisms that enable them to colonize host ...plants and, in some cases, enhance their fitness. In this study, we describe the genomic features predicted to be associated with plant growth-promoting traits in six bacterial communities isolated from sugarcane. The use of highly accurate single-molecule real-time sequencing technology for metagenomic samples from these bacterial communities allowed us to recover 17 genomes. The taxonomic assignments for the binned genomes were performed, revealing taxa distributed across three main phyla: Bacillota, Bacteroidota, and Pseudomonadota, with the latter being the most representative. Subsequently, we functionally annotated the metagenome-assembled genomes (MAGs) to characterize their metabolic pathways related to plant growth-promoting traits. Our study successfully identified the enrichment of important functions related to phosphate and potassium acquisition, modulation of phytohormones, and mechanisms for coping with abiotic stress. These findings could be linked to the robust colonization of these sugarcane endophytes.
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•The genome assembly approach yielded five finished bacterial genomes from metagenomes.•Uncovered new genomes and genes linked to plant growth-promoting traits (PGPTs).•New insights into the complex symbiotic interactions between plants and microorganisms.•Functional complementarity found among plant growth-promoting bacteria.•The Achromobacter genome exhibited the most diverse range of PGPTs.
•We inoculated eight corn fields with an arbuscular mycorrhizal fungus.•Abundance of native mycorrhizal fungi was negatively related to soil nutrient contents.•Competitive effects to native fungal ...communities determined establishment success of the inoculant.•Phosphorous modulated the competitive effects between native and introduced fungi.•Crop growth response to inoculation was negatively correlated to fertilized amounts of P.
A major strategy to increase the sustainability of agricultural systems consists of enhancing internal ecosystem processes that support crop production and reduce external resource inputs. However, specific approaches to achieve this goal still need to be identified. Here, we investigated whether inoculation with a high dose of a well-characterized strain of a plant symbiotic arbuscular mycorrhizal (AM) fungus into Swiss corn fields leads to successful establishment of the fungus in plant roots and can generate agronomic benefits for maize production.
We used single-molecule real-time (SMRT) DNA sequencing to assess community composition of native AM fungi and identified environmental management and biological factors affecting AM fungal abundance, establishment success of the introduced fungus and effects of AMF inoculation on corn yield.
While native AM fungal abundance was negatively related to soil P contents, we found significantly positive relationships between soil P contents and establishment success of the inoculated fungus. There was a significantly negative relationship between inoculum establishment and abundance of native AM fungi. Although molecular quantification using strain-specific qPCR indicated that the inoculated strain strongly increased in abundance in roots from most soils investigated, total AM fungal root colonization was only significantly increased in one soil, indicating successful competition of the inoculant for root niche space against native AM fungi. Positive effects on corn yield were only observed when inoculation increased root colonization and were negatively correlated to P fertilization levels.
The results imply that phosphorus plays a major role in defining the abundance of native AM fungi and the composition of their communities and that these effects can determine establishment success of the inoculant. The results further indicate that positive effects on crop yield may only be expected when potentially achievable root colonization levels are not yet reached and AMF communities are not well developed.
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