PacBio Research Articles

Viral oncogenes, viruses, and cancer: a third-generation sequencing perspective on viral integration into the human genome.

The link between viruses and cancer has intrigued scientists for decades. Certain viruses have been shown to be vital in the development of various cancers by integrating viral DNA into the host genome and activating viral oncogenes. These viruses include the Human Papillomavirus (HPV), Hepatitis B and C Viruses (HBV and HCV), Epstein-Barr Virus (EBV), and Human T-Cell Leukemia Virus (HTLV-1), which are all linked to the development of a myriad of human cancers. Third-generation sequencing technologies have revolutionized our ability to study viral integration events at unprecedented resolution in recent years. They offer long sequencing capabilities along with the ability to map viral integration sites, assess host gene expression, and track clonal evolution in cancer cells. Recently, researchers have been exploring the application of Oxford Nanopore Technologies (ONT) nanopore sequencing and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) sequencing in cancer research. As viral integration is crucial to the development of cancer via viruses, third-generation sequencing would provide a novel approach to studying the relationship interlinking viral oncogenes, viruses, and cancer. This review article explores the molecular mechanisms underlying viral oncogenesis, the role of viruses in cancer development, and the impact of third-generation sequencing on our understanding of viral integration into the human genome.

Benchmarking long-read genome sequence alignment tools for human genomics applications.

The utility of long-read genome sequencing platforms has been shown in many fields including whole genome assembly, metagenomics, and amplicon sequencing. Less clear is the applicability of long reads to reference-guided human genomics, which is the foundation of genomic medicine. Here, we benchmark available platform-agnostic alignment tools on datasets from nanopore and single-molecule real-time platforms to understand their suitability in producing a genome representation. For this study, we leveraged publicly-available data from sample NA12878 generated on Oxford Nanopore and sample NA24385 on Pacific Biosciences platforms. We employed state of the art sequence alignment tools including GraphMap2, long-read aligner (LRA), Minimap2, CoNvex Gap-cost alignMents for Long Reads (NGMLR), and Winnowmap2. Minimap2 and Winnowmap2 were computationally lightweight enough for use at scale, while GraphMap2 was not. NGMLR took a long time and required many resources, but produced alignments each time. LRA was fast, but only worked on Pacific Biosciences data. Each tool widely disagreed on which reads to leave unaligned, affecting the end genome coverage and the number of discoverable breakpoints. No alignment tool independently resolved all large structural variants (1,001-100,000 base pairs) present in the Database of Genome Variants (DGV) for sample NA12878 or the truthset for NA24385. These results suggest a combined approach is needed for LRS alignments for human genomics. Specifically, leveraging alignments from three tools will be more effective in generating a complete picture of genomic variability. It should be best practice to use an analysis pipeline that generates alignments with both Minimap2 and Winnowmap2 as they are lightweight and yield different views of the genome. Depending on the question at hand, the data available, and the time constraints, NGMLR and LRA are good options for a third tool. If computational resources and time are not a factor for a given case or experiment, NGMLR will provide another view, and another chance to resolve a case. LRA, while fast, did not work on the nanopore data for our cluster, but PacBio results were promising in that those computations completed faster than Minimap2. Due to its significant burden on computational resources and slow run time, Graphmap2 is not an ideal tool for exploration of a whole human genome generated on a long-read sequencing platform.

Beyond the exome: utility of long-read whole genome sequencing in exome-negative autosomal recessive diseases

BackgroundLong-read whole genome sequencing (lrWGS) has the potential to address the technical limitations of exome sequencing in ways not possible by short-read WGS. However, its utility in autosomal recessive Mendelian diseases is largely unknown.MethodsIn a cohort of 34 families in which the suspected autosomal recessive diseases remained undiagnosed by exome sequencing, lrWGS was performed on the Pacific Bioscience Sequel IIe platform.ResultsLikely causal variants were identified in 13 (38%) of the cohort. These include (1) a homozygous splicing SV in TYMS as a novel candidate gene for lethal neonatal lactic acidosis, (2) a homozygous non-coding SV that we propose impacts STK25 expression and causes a novel neurodevelopmental disorder, (3) a compound heterozygous SV in RP1L1 with complex inheritance pattern in a family with inherited retinal disease, (4) homozygous deep intronic variants in LEMD2 and SNAP91 as novel candidate genes for neurodevelopmental disorders in two families, and (5) a promoter SNV in SLC4A4 causing non-syndromic band keratopathy. Surprisingly, we also encountered causal variants that could have been identified by short-read exome sequencing in 7 families. The latter highlight scenarios that are especially challenging at the interpretation level.ConclusionsOur data highlight the continued need to address the interpretation challenges in parallel with efforts to improve the sequencing technology itself. We propose a path forward for the implementation of lrWGS sequencing in the setting of autosomal recessive diseases in a way that maximizes its utility.

Reference genome of Townsend's big-eared bat, Corynorhinus townsendii.

Townsend's big-eared bat, Corynorhinus townsendii, is a cave- and mine-roosting species found largely in western North America. Considered a species of conservation concern throughout much of its range, protection efforts would greatly benefit from understanding patterns of population structure, genetic diversity, and local adaptation. To facilitate such research, we present the first de novo genome assembly of C. townsendii as part of the California Conservation Genomics Project (CCGP). Pacific Biosciences HiFi long reads and Omni-C chromatin-proximity sequencing technologies were used to produce a de novo genome assembly, consistent with the standard CCGP reference genome protocol. This assembly comprises 391 scaffolds spanning 2.1 Gb, represented by a scaffold N50 of 174.6 Mb, a contig N50 of 23.4 Mb, and a benchmarking universal single-copy ortholog (BUSCO) completeness score of 96.6%. This high-quality genome will be a key tool for informed conservation and management of this vulnerable species in California and across its range.

Genome assemblies of two species of porcelain crab, Petrolisthes cinctipes and Petrolisthes manimaculis (Anomura: Porcellanidae).

Crabs are a large subtaxon of the Arthropoda, the most diverse and species-rich metazoan group. Several outstanding questions remain regarding crab diversification, including about the genomic capacitors of physiological and morphological adaptation, that cannot be answered with available genomic resources. Physiologically and ecologically diverse Anomuran porcelain crabs offer a valuable model for investigating these questions and hence genomic resources of these crabs would be particularly useful. Here, we present the first two genome assemblies of congeneric and sympatric Anomuran porcelain crabs, Petrolisthes cinctipes and Petrolisthes manimaculis from different microhabitats. Pacific Biosciences high-fidelity sequencing led to genome assemblies of 1.5 and 0.9 Gb, with N50s of 706.7 and 218.9 Kb, respectively. Their assembly length difference can largely be attributed to the different levels of interspersed repeats in their assemblies: The larger genome of P. cinctipes has more repeats (1.12 Gb) than the smaller genome of P. manimaculis (0.54 Gb). For obtaining high-quality annotations of 44,543 and 40,315 protein-coding genes in P. cinctipes and P. manimaculis, respectively, we used RNA-seq as part of a larger annotation pipeline. Contrarily to the large-scale differences in repeat content, divergence levels between the two species as estimated from orthologous protein-coding genes are moderate. These two high-quality genome assemblies allow future studies to examine the role of environmental regulation of gene expression in the two focal species to better understand physiological response to climate change, and provide the foundation for studies in fine-scale genome evolution and diversification of crabs.

Isoform characterisation & splicing signatures of AD‐risk genes using long‐read sequencing

AbstractBackgroundAn increasing number of studies implicate a role for alternative splicing in development and neuropathology of Alzheimer’s disease (AD). However, it has been historically challenging to characterise splicing events, due to the inherent limitations of traditional RNA‐sequencing (RNA‐Seq) to capture full‐length transcripts critical for transcriptome assembly. In this study, we use two complementary targeted long‐read sequencing approaches, Pacific Biosciences (PacBio) isoform sequencing (Iso‐Seq) and Oxford Nanopore Technologies (ONT) nanopore cDNA sequencing, to profile the cortex of a mouse model of tau pathology (rTg4510) with ultra‐deep sequencing of a panel of 20 genes robustly implicated in AD.MethodTargeted PacBio Iso‐Seq and targeted ONT profiling were performed on RNA isolated from 24 female rTg4510 transgenic and wild‐type mice, aged 2, 4, 6 and 8 months. Following successful enrichment for target genes using custom‐designed probes (IDT), library preparation were performed for subsequent sequencing on Sequel and MinION. The panel of genes selected for enrichment included genes (i.e. App, Mapt, Snca) and dementia risk genes identified from GWAS analyses. Raw sequence data was processed using a customised bioinformatics, and full‐length reads were merged and annotated with our developed custom scripts (FICLE).ResultWe identify thousands of novel transcripts across the panel of 20 AD‐ and dementia‐associated genes. We reveal unprecedented diversity of alternatively‐spliced isoforms with widespread usage of alternative novel 5’ and 3’ splice sites. We further robust transcriptional and splicing differences in these AD‐risk genes associated with the development of tau pathology. Among these changes, we observed global up‐regulation of Trem2‐associated isoforms and isoform switches in Bin1, further supporting a role for the dysregulation of the immune response in the development of AD pathology.ConclusionThis represents the first study to comprehensively assess variable AS associated with the development of tau pathology using both PacBio Iso‐Seq and ONT nanopore sequencing. Our analyses confirm the importance of alternative RNA splicing in AD and identify evidence of differential transcript usage, even in the absence of gene‐level expression alterations. Further work will be undertaken to validate and functionally characterise these novel isoforms, and to extend these analyses to human post‐mortem AD brain samples.

Whole-genome long-read sequencing downsampling and its effect on variant-calling precision and recall.

Advances in long-read sequencing (LRS) technologies continue to make whole-genome sequencing more complete, affordable, and accurate. LRS provides significant advantages over short-read sequencing approaches, including phased de novo genome assembly, access to previously excluded genomic regions, and discovery of more complex structural variants (SVs) associated with disease. Limitations remain with respect to cost, scalability, and platform-dependent read accuracy and the tradeoffs between sequence coverage and sensitivity of variant discovery are important experimental considerations for the application of LRS. We compare the genetic variant-calling precision and recall of Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) HiFi platforms over a range of sequence coverages. For read-based applications, LRS sensitivity begins to plateau around 12-fold coverage with a majority of variants called with reasonable accuracy (F1 score above 0.5), and both platforms perform well for SV detection. Genome assembly increases variant-calling precision and recall of SVs and indels in HiFi data sets with HiFi outperforming ONT in quality as measured by the F1 score of assembly-based variant call sets. While both technologies continue to evolve, our work offers guidance to design cost-effective experimental strategies that do not compromise on discovering novel biology.

#201 : Comparison of the Sensitivity of Detecting Cervical Bacteria with Next Generation Sequencing and Third Generation Sequencing Technologies

Background and Aims: In in vitro fertilization (IVF) cycles, some patients suffering with vaginosis showed poor reproductive outcome even transferring with good quality embryos. The aim of this pilot study was to evaluate whether the species of Lactobacillus could be detected by next generation sequencing (NGS) and Third generation sequencing (TGS). Method: 18 Patients aged 32–48 years-old who visited our fertility center from February 2021 to June 2022 with previous failure of transfer cycle were included. Exclusion criteria was antibiotic treatment within 3 months prior to enrolment. Genomic DNA of cervical microbiota taken from Cervico vaginal swabs in all patients were extracted and amplified. NGS was performed following the protocol of Ion 16S Metagenomics Kit by detecting V2–4–8 and V3–6, 7–9 regions of 16S. Amplicons were sequenced with Ion GeneStudio S5 Prime System. For TGS, full length of 16S was sequenced with Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences) and analyzed. Results: All of the 18 cervical samples could be amplified with V2–4–8 and V3–6, 7–9 primers and the genus could be 100% identified by NGS. However, most of the Lactobacillus includes L. crispatus, L. jensenii. and L. gasseri showed indistinguishable except of L. iners. On the other hand, TGS clearly identified all Lactobacillus. For Gardnerella, Atopobium and Prevotella, TGS and NGS showed equivalent sensitivity in genus level. However, due to the sequence similarity, Escherichia/Shigella could not be identified from the Lactobacillus. Conclusion: In this report, we aim to compare the sensitivity of detecting bacteria by 16S amplification method. Preliminary results suggest that NGS can distinguish bacterium causes vaginosis in genus level, but there might be misleading if the existence of pathogen such as Escherichia/Shigella. Till now, TGS still exhibits the best sensitivity for distinguish Lactobacillus in species level.

Long‐read sequencing of the whole‐blood compartment reveals similar telomere lengths between Alzheimer’s disease patients and cognitively‐healthy centenarians

AbstractBackgroundTelomere stability may be a potential biomarker for Alzheimer’s disease progression. Indeed, previous studies have observed significant associations with telomere length and risk for Alzheimer’s disease (AD) in both carriers and non‐carriers of APOE4. In contrast, (cognitively healthy) centenarians—an extreme control group in the spectrum of brain health—have been observed to harbour longer telomere lengths relative to younger age groups. In this study, we investigated telomere stability in the whole‐blood compartment of AD patients, centenarians, and centenarian children through single‐molecule long‐read sequencing.MethodLong‐read HiFi sequencing of the whole‐blood compartment was performed in AD patients (n = 59, mean age = 68.5), centenarians (n = 84, mean age = 101.6), and centenarian children (n = 10, mean age = 71.0), using the Pacific Bioscience Sequel II instrument. For each sample, we identified reads with telomeric content based on alignments to the CHM13 reference genome. Per‐read telomere lengths were calculated via a high‐density clustering approach of the TTAGGG repeat sequence, keeping only reads that have a higher likelihood of harbouring complete telomeric content. The sub‐telomeric portion of each read was then used to assign chromosomal origin based on sequence similarity all chromosomes in CHM13. Whole‐blood and chromosome‐specific telomere lengths were then compared across the three sample‐groups.ResultCentenarian children were observed to harbour the longest telomeres with a median length of 6.88 kbp, and 95% C.I. of 6.78‐7.00 kbp. This was followed by AD patients with median length of 5.82 kbp, 95% CI 5.78‐5.85 kb; and centenarians with a median length of 5.52 kbp, and 95% C.I. of 5.50‐5.55 kbp. Telomere lengths across all groups were significantly different (p < 0.001). A similar pattern was observed across each individual chromosome, with the exception of chromosome X which harboured smaller telomere lengths in AD patients and centenarians.Centenarians harboured significantly smaller telomere lengths than their children (p < 0.001), with the exception of two centenarians that did not have significant differences.ConclusionDespite being aged‐match, AD patients harbour shorter telomere lengths than centenarian children, and only slightly longer telomere lengths than centenarians.

Genomics and transcriptomics of the Chinese mitten crabs (Eriocheir sinensis)

To gain a deeper understanding of the genetic factors influencing the growth and development of Eriocheir sinensis, a well-known species of hairy crab found in Yangcheng Lake, this study focused on the de novo genome and full-length transcriptome information of the selected subjects. Specifically, Yangcheng Lake hairy crabs were chosen as the experimental samples. Initially, a genome analysis was performed, resulting in the identification of gene fragments with a combined length of 1266,092,319 bp. Subsequently, a transcriptome analysis was conducted on a mixture of tissues from four different sites, namely muscle, brain, eye, and heart, to further investigate the genetic characteristics at the transcriptome level. The Pacific Biosciences (Pacio) single-molecule real-time sequencing system generated a total of 36.93 G sub-fragments and 175,90041 effective inserts. This research contributes to the indirect comprehension of genetic variations underlying individual traits. Furthermore, a comparison of the obtained data with relevant literature emphasizes the advantages of this study and establishes a basis for further investigations on the Chinese mitten crab.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Genome Sequencing of Strain Vm263, a Hypotoxic Strain of Valsa mali Causing Apple Valsa Canker

Apple valsa canker, caused by Valsa mali, has seriously endangered the sustainable development of the apple industry in China. Our previous study revealed that low-temperature induction increased the pathogenicity of strain Vm263, which is a hypotoxic strain of V. mali causing apple valsa canker. However, the regulatory mechanism of this phenomenon was still unclear. In this study, whole genome sequencing of strain Vm263 was performed using a combination of long-read single-molecule real-time sequencing technology (Pacific Biosciences) and MGISEQ sequencing. The genome size of strain Vm263 was 42.83 Mb and the assembly N50 was 5.09 Mb. The genome provides a valuable resource for facilitating our understanding of the response of V. mali to low temperature. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Efficient end-to-end long-read sequence mapping using minimap2-fpga integrated with hardware accelerated chaining

minimap2 is the gold-standard software for reference-based sequence mapping in third-generation long-read sequencing. While minimap2 is relatively fast, further speedup is desirable, especially when processing a multitude of large datasets. In this work, we present minimap2-fpga, a hardware-accelerated version of minimap2 that speeds up the mapping process by integrating an FPGA kernel optimised for chaining. Integrating the FPGA kernel into minimap2 posed significant challenges that we solved by accurately predicting the processing time on hardware while considering data transfer overheads, mitigating hardware scheduling overheads in a multi-threaded environment, and optimizing memory management for processing large realistic datasets. We demonstrate speed-ups in end-to-end run-time for data from both Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio). minimap2-fpga is up to 79% and 53% faster than minimap2 for sim 30times ONT and sim 50times PacBio datasets respectively, when mapping without base-level alignment. When mapping with base-level alignment, minimap2-fpga is up to 62% and 10% faster than minimap2 for sim 30times ONT and sim 50times PacBio datasets respectively. The accuracy is near-identical to that of original minimap2 for both ONT and PacBio data, when mapping both with and without base-level alignment. minimap2-fpga is supported on Intel FPGA-based systems (evaluations performed on an on-premise system) and Xilinx FPGA-based systems (evaluations performed on a cloud system). We also provide a well-documented library for the FPGA-accelerated chaining kernel to be used by future researchers developing sequence alignment software with limited hardware background.

Comparative transcriptome analysis of dioecious floral development in Trachycarpus fortunei using Illumina and PacBio SMRT sequencing

BackgroundTrachycarpus fortunei is a plant with significant economic and ornamental value. Both male and female flowers of T. fortunei originate as bisexual flowers, and selective abortion occurs during floral development. However, the regulatory mechanisms underlying this process remain unclear in T. fortunei. In this study, transcriptome sequencing with Illumina and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) platforms were used to investigate gene expression differences between male and female T. fortunei plants.ResultsA total of 833,137 full-length non-chimeric (FLNC) reads were obtained, and 726,846 high-quality full-length transcripts were identified. A total of 159 genes were differentially expressed between male and female flowers at all development stages. Some of the differentially expressed genes (DEGs) showed male bias, including serine/threonine-protein kinase (STPK), THUMP1 homolog and other genes. Through single-nucleotide polymorphisms(SNPs) identification, 28 genes were considered as potential sex-associated SNPs. Time-Ordered Gene Co-expression Network (TO-GCN) analysis revealed that MADS2 and MADS26 may play important roles in the development of female and male flowers T. fortune plants, respectively.ConclusionsThese findings provide a genetic basis for flower development and differentiation in T. fortunei, and improve our understanding of the mechanisms underlying sexual differentiation in T. fortunei.

Novel Double Fusion (βδβ) Hemoglobin Variant with Comparison to Lepore Hemoglobins

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

Transcript Isoform Diversity of Ampliconic Genes on the Y Chromosome of Great Apes.

Y chromosomal ampliconic genes (YAGs) are important for male fertility, as they encode proteins functioning in spermatogenesis. The variation in copy number and expression levels of these multicopy gene families has been studied in great apes; however, the diversity of splicing variants remains unexplored. Here, we deciphered the sequences of polyadenylated transcripts of all nine YAG families (BPY2, CDY, DAZ, HSFY, PRY, RBMY, TSPY, VCY, and XKRY) from testis samples of six great ape species (human, chimpanzee, bonobo, gorilla, Bornean orangutan, and Sumatran orangutan). To achieve this, we enriched YAG transcripts with capture probe hybridization and sequenced them with long (Pacific Biosciences) reads. Our analysis of this data set resulted in several findings. First, we observed evolutionarily conserved alternative splicing patterns for most YAG families except for BPY2 and PRY. Second, our results suggest that BPY2 transcripts and proteins originate from separate genomic regions in bonobo versus human, which is possibly facilitated by acquiring new promoters. Third, our analysis indicates that the PRY gene family, having the highest representation of noncoding transcripts, has been undergoing pseudogenization. Fourth, we have not detected signatures of selection in the five YAG families shared among great apes, even though we identified many species-specific protein-coding transcripts. Fifth, we predicted consensus disorder regions across most gene families and species, which could be used for future investigations of male infertility. Overall, our work illuminates the YAG isoform landscape and provides a genomic resource for future functional studies focusing on infertility phenotypes in humans and critically endangered great apes.

New horizons of human genetics in digestive diseases

Recent studies have decoded the human Y chromosome sequencing with predominant precision and coverage, offering promising prospects in human genetics and clinical translation. Such an achievement is facilitated by third-generation...

Two long read-based genome assembly and annotation of polyploidy woody plants, Hibiscus syriacus L. using PacBio and Nanopore platforms

Improvements in long read DNA sequencing and related techniques facilitated the generation of complex eukaryotic genomes. Despite these advances, the quality of constructed plant reference genomes remains relatively poor due to the large size of genomes, high content of repetitive sequences, and wide variety of ploidy. Here, we developed the de novo sequencing and assembly of high polyploid plant genome, Hibiscus syriacus, a flowering plant species of the Malvaceae family, using the Oxford Nanopore Technologies and Pacific Biosciences Sequel sequencing platforms. We investigated an efficient combination of high-quality and high-molecular-weight DNA isolation procedure and suitable assembler to achieve optimal results using long read sequencing data. We found that abundant ultra-long reads allow for large and complex polyploid plant genome assemblies with great recovery of repetitive sequences and error correction even at relatively low depth Nanopore sequencing data and polishing compared to previous studies. Collectively, our combination provides cost effective methods to improve genome continuity and quality compared to the previously reported reference genome by accessing highly repetitive regions. The application of this combination may enable genetic research and breeding of polyploid crops, thus leading to improvements in crop production.

A chromosome-level genome assembly for Chinese plum ‘Wushancuili’ reveals the molecular basis of its fruit color and susceptibility to rain-cracking

Chinese plum (Prunus salicina Lindl.) originates from China and makes a large contribution to the global production of plums. The P. salicina ‘Wushancuili’ has a green coloration and high fruit quality and is economically important in eliminating poverty and protecting ecology in the Yangtze River Three Gorges Reservoir. However, rain-induced cracking (rain-cracking, literally skin cracking caused by rain) is a limitation to ‘Wushancuili’ fruit production and causes severe losses. This study reported a high-quality ‘Wushancuili’ genome assembly consisting of a 302.17-Mb sequence with eight pseudo-chromosomes and a contig N50 of 23.59 Mb through the combination of Illumina sequencing, Pacific Biosciences HiFi Ⅲ sequencing, and high-throughput chromosome conformation capture technology. A total of 25 109 protein-coding genes are predicted and 54.17% of the genome is composed of repetitive sequences. ‘Wushancuili’ underwent a remarkable orthoselection during evolution. Gene identification revealed that loss-of-function in four core MYB10 genes results in the anthocyanin deficiency and absence of red color, revealing the green coloration due to the residual high chlorophyll in fruit skin. Besides, the occurrence of cracking is assumed to be closely associated with cell wall modification and frequently rain-induced pathogen enrichment through transcriptomic analysis. The loss of MYB10 genes might render fruit more susceptible to pathogen-mediated cracking by weakening the epidermal strength and reactive oxygen species (ROS) scavenging. Our findings provided fundamental knowledge regarding fruit coloration and rain-cracking and will facilitate genetic improvement and cultivation management in Chinese plums.

Single-Molecule Poly(A) Tail Sequencing (SM-PATseq) Using the PacBio Platform.

The polyadenylation of the 3' ends of messenger RNAs is an important regulator of stability and translation. We developed the single-molecule poly(A) tail sequencing method, SM-PATseq, to assay tail lengths of the whole transcriptome at nucleotide resolution using long-read sequencing. This method generates cDNA using an oligo-dT 3' splint adaptor ligation to prime first-strand cDNA synthesis, followed by random hexamer priming for second-strand synthesis. By directly sequencing the cDNA on long-read platforms, we can resolve tail lengths at nucleotide resolution, identify non-A bases within the tail, and quantify transcript abundance analogous to traditional RNAseq methods. Here, we discuss the method for generating, sequencing, and primary analysis of poly(A) tail data from total RNA using the Pacific Biosciences Sequel platform.