PacBio Sequencing Research Articles

Chromosome-scale genome of the human blood fluke Schistosoma mekongi and its implications for public health.

Schistosoma mekongi is a human blood fluke causing schistosomiasis that threatens approximately 1.5 million humans in the world. Nonetheless, the limited available S. mekongi genomic resources have hindered understanding of its biology and parasite-host interactions for disease management and pathogen control. The aim of our study was to integrate multiple technologies to construct a high-quality chromosome-level assembly of the S. mekongi genome. The reference genome for S. mekongi was generated through integrating Illumina, PacBio sequencing, 10 × Genomics linked-read sequencing, and high-throughput chromosome conformation capture (Hi-C) methods. In this study, we conducted de novo assembly, alignment, and gene prediction to assemble and annotate the genome. Comparative genomics allowed us to compare genomes across different species, shedding light on conserved regions and evolutionary relationships. Additionally, our transcriptomic analysis focused on genes associated with parasite-snail interactions in S. mekongi infection. We employed gene ontology (GO) enrichment analysis for functional annotation of these genes. In the present study, the S. mekongi genome was both assembled into 8 pseudochromosomes with a length of 404Mb, with contig N50 and scaffold N50 lengths of 1168kb and 46,759kb, respectively. We detected that 43% of the genome consists of repeat sequences and predicted 9103 protein-coding genes. We also focused on proteases, particularly leishmanolysin-like metalloproteases (M8), which are crucial in the invasion of hosts by 12 flatworm species. Through phylogenetic analysis, it was discovered that the M8 gene exhibits lineage-specific amplification among the genus Schistosoma. Lineage-specific expansion of M8 was observed in blood flukes. Additionally, the results of the RNA-seq revealed that a mass of genes related to metabolic and biosynthetic processes were up-regulated, which might be beneficial for cercaria production. This study delivers a high-quality, chromosome-scale reference genome of S. mekongi, enhancing our understanding of the divergence and evolution of Schistosoma. The molecular research conducted here also plays a pivotal role in drug discovery and vaccine development. Furthermore, our work greatly advances the understanding of host-parasite interactions, providing crucial insights for schistosomiasis intervention strategies.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer

Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 were not previously reported. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-β/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine.

The gene cluster associated with strong biofilm-formation capacity by histamine-producing Lentilactobacillus parabuchneri encodes a sortase-mediated pilus and is located on a plasmid

Histamine is a biogenic amine synthesized through the enzymatic decarboxylation of the amino acid histidine. It can accumulate at high concentrations in foods through the metabolism of certain bacteria, sometimes leading to adverse reactions in consumers. In cheese, histamine can accumulate at toxic levels; Lentilactobacillus parabuchneri has been identified the major cause of this problem. Previous studies have shown some L. parabuchneri strains to form biofilms on different surfaces, posing a contamination risk during cheese production, particularly for cheeses that are processed post-ripening (e.g., grating or slicing). The food contamination they cause can result in economic losses and even foodborne illness if histamine accumulates in the final product. The aim of the present work was to identify the genes of L. parabuchneri involved in biofilm formation, and to determine their function. The genomes of six strains with different biofilm-production capacities (strong, moderate and weak) were sequenced and analysed. A cluster of four genes , similar to those involved in sortase-mediated pilus formation, was identified in the strong biofilm-producers, suggesting it to have a role in surface adhesion. Cloning and heterologous expression in Lactococcus cremoris NZ9000 confirmed its functionality and involvement in adhesion and, therefore, in biofilm formation. PacBio sequencing showed this cluster to be located on a 33.4 kb plasmid, which might increase its chances of horizontal transmission. These findings provide insight into the genetic factors associated with biofilm formation in histamine-producing L. parabuchneri, and into the risks associated with this bacterium in cheese production.

Chromosome-level genome assembly of Przevalski’s partridge (Alectoris magna)

Przevalski’s partridge (Alectoris magna) is one of the birds in the genus Alectoris endemic to China. The distribution of A. magna was narrow, and it was only found in parts of the Qinghai, Gansu, and Ningxia provinces. A. magna was considered a monotypic species until it was distinguished into two subspecies. However, external morphological characteristics, rather than genetic differences or evolutionary relationships, are now commonly used as evidence of subspecies differentiation. In this study, a chromosome-level reference genome of A. magna has been constructed by combining Illumina, PacBio and Hi-C sequencing data. The 1135.01 Mb A. magna genome was ultimately assembled. The genome showed 96.9% completeness (BUSCO), with a contig N50 length of 23.34 Mb. The contigs were clustered and oriented on 20 chromosomes, covering approximately 99.96% of the genome assembly. Additionally, altogether 19,103 protein-coding genes were predicted, of which 95.10% were functionally annotated. This high-quality genome assembly could serve as a valuable genomic resource for future research on the functional genomics, genetic protection, and interspecific hybridization of A. magna.

An improved reference genome and first organelle genomes of Quercus suber

Cork oak (Quercus suber L.) is an ecologically and economically important evergreen tree species native to the Mediterranean region and widespread in southwest Europe and northwest Africa. An improved genome assembly of cork oak using a combination of Illumina and PacBio sequencing is presented in this study. The assembled genome contains 2351 scaffolds longer than 1000 bp, accounting for 765.7 Mbp of genome size, L90 of 755, and a N50 of 1.0 Mbp, with 40,131 annotated genes. The repetitive sequences constitute 53.6% of the genome. The genome sequences of chloroplast and mitochondrion were determined for the first time, with a genome size of 161,179 bp and 531,858 bp, respectively. Phylogenetic analysis based on complete chloroplast genome sequence showed that Q. suber is closely related to Quercus variabilis, two cork-producing species with commercial use. All data generated are available through the public databases, being ready to be used without restrictions. This study provides an improved nuclear genome assembly together with the organelle genomes of cork oak. These resources will be useful for further breeding strategies and conservation programs and for comparative genomic studies in oak species.

Transcriptome analysis of Lantana camara flower petals reveals candidate anthocyanin biosynthesis genes mediating red flower color development.

Flower color plays a crucial role in the appeal and selection of ornamental plants, directly influencing breeding strategies and the broader horticulture industry. Lantana camara, a widely favored flowering shrub, presents a rich palette of flower colors. Yet, the intricate molecular mechanisms governing this color variation in the species have remained largely unidentified. With the aim of filling this gap, this study embarked on a comprehensive de novo transcriptome assembly and differential gene expression analysis across 3 distinct lantana accessions, each showcasing a unique flower color. By harnessing the capabilities of both PacBio and Illumina sequencing platforms, a robust transcriptome assembly, encompassing 123,492 gene clusters and boasting 94.2% BUSCO completeness, was developed. The differential expression analysis unveiled 72,862 unique gene clusters that exhibited varied expression across different flower stages. A pronounced upregulation of 8 candidate core anthocyanin biosynthesis genes in the red-flowered accession was uncovered. This was further complemented by an upregulation of candidate MYB75 (PAP1) and bHLH42 (TT8) transcription factors. A candidate carotenoid cleavage dioxygenase (CCD4a) gene cluster also manifested a marked upregulation in white flowers. The study unveils the molecular groundwork of lantana's flower color variation, offering insights for future research and potential applications in breeding ornamental plants with desired color traits.

PacBio Sequencing Reveals Microbial Community Diversity in Man-made and Natural Habitats

PacBio Sequencing Reveals Microbial Community Diversity in Man-made and Natural Habitats

Genome Assembly and Microsatellite Marker Development Using Illumina and PacBio Sequencing in the Carex pumila (Cyperaceae) from Korea.

This study is the first to report the characterization of Carex pumila genomic information. Assembly of the genome generated a draft of C. pumila based on PacBio Sequel II and Illumina paired-end sequencing, which was assembled from 2941 contigs with an estimated genome size of 0.346 Gb. The estimate of repeats in the genome was 31.0%, and heterozygosity ranged from 0.426 to 0.441%. The integrity evaluation of the assembly revealed 1481 complete benchmarked universal single-copy orthologs (BUSCO) (91.76%), indicating the high quality of the draft assembly. A total of 23,402 protein-coding genes were successfully predicted and annotated in the protein database. UpsetR plots showed that 7481 orthogroups were shared by all species. The phylogenetic tree showed that C. pumila is a close but distant relative of Ananas comosus. C. pumila had greater contraction (3154) than expansion (392). Among the extended gene families, aquaporins have been found to be enriched. Primers for microsatellite markers determined 30 polymorphic markers out of 100. The average number of alleles amplified by these 30 polymorphic markers was 4 to 12, with an average polymorphism information content (PIC) value of 0.660. In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of C. pumila.

Genome Sequence Resource for Phytopythium helicoides, the Causal Pathogen of Crown and Root Rot of Photinia × fraseri.

The oomycete Phytopythium helicoides is a globally distributed plant pathogen, and causes crown blight, root rot and seedling damping in many agricultural plants. P. helicoides PF-he2 was isolated from infected Photinia × fraseri Dress in China. Here, a high-quality genome of PF-he2 was sequenced by a combination of PacBio and Illumina sequencing strategies. The genome is 49.09 Mb in length with 105 contigs. The N50 contig length is 860 kb, and BUSCO completeness is 94%. Gene prediction resulted in 16,807 protein-coding genes, and 1,663 secreted proteins were identified. In addition, we identified a series of proteins involved in pathogenicity, including 30 CRN effectors, 26 YxSL[RK] effectors, 30 NLP proteins, and 49 elicitin-like proteins. This genome provides a valuable resource for better understanding the genetic diversity and molecular basis of pathogenesis of P. helicoides, which will facilitate the development of effective control strategies.

Improving the RNA velocity approach with single-cell RNA lifecycle (nascent, mature and degrading RNAs) sequencing technologies.

We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and degrading RNAs) in cells. Additionally, we updated our HIT-scISOseq V2 to discover a more accurate RNA lifecycle using 10x Chromium and Pacbio sequencing. Most importantly, to explore how single-cell full-length RNA sequencing technologies could help improve the RNA velocity approach, we introduced a new algorithm called 'Region Velocity' to more accurately configure cellular RNA velocity. We applied this algorithm to study spermiogenesis and compared the performance of FLOUR-seq with Pacbio-based HIT-scISOseq V2. Our findings demonstrated that 'Region Velocity' is more suitable for analyzing single-cell full-length RNA data than traditional RNA velocity approaches. These novel methods could be useful for researchers looking to discover full-length RNAs in single cells and comprehensively monitor RNA lifecycle in cells.

PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages.

Gut phages have an important impact on human health. Methylation plays key roles in DNA recognition, gene expression regulation and replication for phages. However, the DNA methylation landscape of gut phages is largely unknown. Here, with PacBio sequencing (2120×, 4785 Gb), we detected gut phage methylation landscape based on 22673 gut phage genomes, and presented diverse methylation motifs and methylation differences in genomic elements. Moreover, the methylation rate of phages was associated with taxonomy and host, and N6-methyladenine methylation rate was higher in temperate phages than in virulent phages, suggesting an important role for methylation in phage-host interaction. In particular, 3543 (15.63%) phage genomes contained restriction-modification system, which could aid in evading clearance by the host. This study revealed the DNA methylation landscape of gut phage and its potential roles, which will advance the understanding of gut phage survival and human health.

JAK2V617F mutation and associated chromosomal alterations in primary and secondary myelofibrosis and post-HCT outcomes

AbstractJAK2V617F is the most common driver mutation in primary or secondary myelofibrosis for which allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment. Knowledge of the prognostic utility of JAK2 alterations in the HCT setting is limited. We identified all patients with MF who received HCT between 2000 and 2016 and had a pre-HCT blood sample (N = 973) available at the Center of International Blood and Marrow Transplant Research biorepository. PacBio sequencing and single nucleotide polymorphism–array genotyping were used to identify JAK2V617F mutation and associated mosaic chromosomal alterations (mCAs), respectively. Cox proportional hazard models were used for HCT outcome analyses. Genomic testing was complete for 924 patients with MF (634 primary MF [PMF], 135 postpolycythemia vera [PPV-MF], and 155 postessential thrombocytopenia [PET-MF]). JAK2V617F affected 562 patients (57.6% of PMF, 97% of PPV-MF, and 42.6% of PET-MF). Almost all patients with mCAs involving the JAK2 region (97.9%) were JAK2V617-positive. In PMF, JAK2V617F mutation status, allele burden, or identified mCAs were not associated with disease progression/relapse, nonrelapse mortality (NRM), or overall survival. Almost all PPV-MF were JAK2V617F-positive (97%), with no association between HCT outcomes and mutation allele burden or mCAs. In PET-MF, JAK2V617F high mutation allele burden (≥60%) was associated with excess risk of NRM, restricted to transplants received in the era of JAK inhibitors (2013-2016; hazard ratio = 7.65; 95% confidence interval = 2.10-27.82; P = .002). However, allele burden was not associated with post-HCT disease progression/relapse or survival. Our findings support the concept that HCT can mitigate the known negative effect of JAK2V617F in patients with MF, particularly for PMF and PPV-MF.

The first chromosome-level genome assembly and transcriptome sequencing provide insights into cantharidin production of the blister beetles.

Blister beetles (Coleoptera: Meloidae) produce a natural defensive toxin cantharidin (CTD), which has been used for various cancer treatments and other diseases. Currently, the lack of chromosome-level reference genomes in Meloidae limits further understanding of the mechanism of CTD biosynthesis and environmental adaptation. In this study, the chromosome-level genome assembly of Mylabris phalerata was generated based on PacBio and Hi-C sequencing. This reference genome was about 136.68 Mb in size with contig N50 of 9.17 Mb and composed of 12 chromosomes. In comparison to six other Coleoptera insects, M. phalerata exhibited multiple expanded gene families enriched in juvenile hormone (JH) biosynthetic process pathway, farnesol dehydrogenase activity, and cytochrome P450, which may be related to CTD biosynthesis. Consistently, the transcriptomic analysis suggested the "terpenoid backbone biosynthesis" pathway and "the juvenile hormone" as putative core pathways of CTD biosynthesis and presented eight up-regulated differential expression genes in male adults as candidate genes. It is possible that the restricted feeding niche and lifestyle of M. phalerata were the cause of the gene family's contraction of odorant binding proteins. The ABC transporters (ABCs) related to exporting bound toxins out of the cell and the resistance to the self-secreted toxins (e.g. CTD) were also contracted, possibly due to other self-protection strategies in M. phalerata. A foundation of understanding CTD biosynthesis and environmental adaptation of blister beetles will be established by our reference genome and discoveries.

Complete Mitochondrial Genome Assembly of an Upland Wild Rice Species, Oryza granulata and Comparative Mitochondrial Genomic Analyses of the Genus Oryza.

Wild upland rice species, including Oryza granulata, possess unique characteristics that distinguish them from other Oryza species. For instance, O. granulata characteristically has a GG genome and is accordingly classified as a basal lineage of the genus Oryza. Here, we deployed a versatile hybrid approach by integrating Illumina and PacBio sequencing data to generate a high-quality mitochondrial genome (mitogenome) assembly for O. granulata. The mitogenome of O. granulata was 509,311 base pairs (bp) with sixty-seven genes comprising two circular chromosomes, five ribosomal RNA (rRNA) coding genes, twenty-five transfer RNA (tRNA) coding genes, and thirty-seven genes coding for proteins. We identified a total of 378 simple sequence repeats (SSRs). The genome also contained 643 pairs of dispersed repeats comprising 340 palindromic and 303 forward. In the O. granulata mitogenome, the length of 57 homologous fragments in the chloroplast genome occupied 5.96% of the mitogenome length. Collinearity analysis of three Oryza mitogenomes revealed high structural variability and frequent rearrangements. Phylogenetic analysis showed that, compared to other related genera, O. granulata had the closest genetic relationship with mitogenomes reported for all members of Oryza, and occupies a position at the base of the Oryza phylogeny. Comparative analysis of complete mitochondrial genome assemblies for Oryza species revealed high levels of mitogenomic diversity, providing a foundation for future conservation and utilization of wild rice biodiversity.

Complete Genome Resource of a Hypervirulent Xanthomonas oryzae pv. oryzae Strain YNCX Isolated from Yunnan Plateau Japonica Rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight (BLB), is one of the most destructive bacterial pathogens in rice production worldwide. Although several complete genome sequences of Xoo strains have been released in public databases, they are mainly isolated from low-altitude indica rice cultivating areas. Here, a hypervirulent strain, YNCX, isolated from the high-altitude japonica rice-growing region in Yunnan Plateau, was used to extract genomic DNA for PacBio sequencing and Illumina sequencing. After assembly, a high-quality complete genome consisting of a circular chromosome and six plasmids was generated. The genome sequence of YNCX provides a valuable resource for high-altitude races and enables the identification of new virulence TALE effectors, contributing to a better understanding of rice-Xoo interactions.

Mucosal Microbiome in Patients with Early Bowel Polyps: Inferences from Short-Read and Long-Read 16S rRNA Sequencing.

Numerous studies have correlated dysbiosis in stool microbiota with colorectal cancer (CRC); however, fewer studies have investigated the mucosal microbiome in pre-cancerous bowel polyps. The short-read sequencing of variable regions in the 16S rRNA gene has commonly been used to infer bacterial taxonomy, and this has led, in part, to inconsistent findings between studies. Here, we examined mucosal microbiota from patients who presented with one or more polyps, compared to patients with no polyps, at the time of colonoscopy. We evaluated the results obtained using both short-read and PacBio long-read 16S rRNA sequencing. Neither sequencing technology identified significant differences in microbial diversity measures between patients with or without bowel polyps. Differential abundance measures showed that amplicon sequence variants (ASVs) associated with Ruminococcus gnavus and Escherichia coli were elevated in mucosa from polyp patients, while ASVs associated with Parabacteroides merdae, Veillonella nakazawae, and Sutterella wadsworthensis were relatively decreased. Only R. gnavus was consistently identified using both sequencing technologies as being altered between patients with polyps compared to patients without polyps, suggesting differences in technologies and bioinformatics processing impact study findings. Several of the differentially abundant bacteria identified using either sequencing technology are associated with inflammatory bowel diseases despite these patients being excluded from the current study, which suggests that early bowel neoplasia may be associated with a local inflammatory niche.

Development and Genome-Wide Analysis of a Blast-Resistant japonica Rice Variety.

Rice is one of the most important crops in the world, and its production is severely affected by the rice blast disease caused by the fungus Magnaporthe oryzae. Several major blast resistance genes and QTLs associated with blast resistance have been described and mostly identified in indica rice varieties. In this work, we report the obtention of a blast-resistant rice breeding line derived from crosses between the resistant indica variety CT13432 and the japonica elite cultivar JSendra (highly susceptible to blast). The breeding line, named COPSEMAR9, was found to exhibit resistance to leaf blast and panicle blast, as demonstrated by disease assays under controlled and field conditions. Furthermore, a high-quality genome sequence of the blast-resistant breeding line was obtained using a strategy that combines short-read sequencing (Illumina sequencing) and long-read sequencing (Pacbio sequencing). The use of a whole-genome approach allowed the fine mapping of DNA regions of indica and japonica origin present in the COPSEMAR9 genome and the identification of parental gene regions potentially contributing to blast resistance in the breeding line. Rice blast resistance genes (including Pi33 derived from the resistant parent) and defense-related genes in the genome of COPSEMAR9 were identified. Whole-genome analyses also revealed the presence of microRNAs (miRNAs) with a known function in the rice response to M. oryzae infection in COPSEMAR9, which might also contribute to its phenotype of blast resistance. From this study, the genomic information and analysis methods provide valuable knowledge that will be useful in breeding programs for blast resistance in japonica rice cultivars.

Whole-genome sequence of Staphylococcus epidermidis strain AH3, isolated from Nile tilapia (Oreochromis niloticus).

The whole-genome sequence of Staphylococcus epidermidis strain AH3 isolated from moribund farmed Nile tilapia (Oreochromis niloticus) was performed using a combination of the Illumina and Pacific Biosciences (PacBio) sequencing platforms. The genome sequence is composed of a single chromosome of 2,464,380 bp with a GC content of 32.2% and 2,220 predicted protein-coding genes.

Anatomical and Transcriptome Analyses of Moso Bamboo Culm Neck Growth: Unveiling Key Insights.

The Moso bamboo culm neck, connected with the rhizome and the shoot bud, is an important hub for connecting and transporting the aboveground and belowground systems of bamboo for the shoot bud development and rapid growth. Our previous study revealed that the culm neck generally undergoes six different developmental stages (CNS1-CNS6), according to the primary thickening growth of the underground shoot bud. However, the molecular mechanism of the culm neck development remains unknown. The present study focused on the developmental process of the CNS3-CNS5 stages, representing the early, middle, and late elongation stages, respectively. These stages are densely packed with vascular tissues and consist of epidermis, hypodermis, cortex, and ground tissue. Unlike the hollow structure of the culms, the culm necks are solid structures. As the culm neck continues to grow, the lignin deposition increases noticeably, contributing to its progressive strengthening. For the transcriptome analysis, a total of 161,160 transcripts with an average length of 2373 were obtained from these stages using both PacBio and Illumina sequencing. A total of 92.2% of the reads mapped to the Moso bamboo reference genome. Further analysis identified a total of 5524 novel genes and revealed a dynamic transcriptome. Secondary-metabolism- and transport-related genes were upregulated particularly with the growth of the culm neck. Further analysis revealed the molecular processes of lignin accumulation in the culm neck, which include differentially expressed genes (DEGs) related to cell wall loosening and remodeling and secondary metabolism. Moreover, the upregulations of transcription factors such as MYBH and RSM in the MYB family play crucial roles during critical transitions in the culm neck development, such as changes in the angle between the rhizome and the culm neck. Our new findings provide essential insights into the cellular roadmaps, transcriptional networks, and key genes involved in the culm neck development.