Third-generation Long-read Sequencing Research Articles

Detecting serologically difficult ABO blood groups using single-molecule real-time sequencing technology.

Recently, third-generation long-read sequencing technology has been increasingly applied to the detection of various blood group systems. Because of its long read length and use of single-molecule sequencing, it is capable of obtaining the sequences of blood group genes in their entirety as well as of distinguishing haplotypes. Therefore, here, we collected ABO blood group samples that were difficult to classify serologically and analysed the sequences of the coding regions of the ABO genes as well as the sequences upstream and downstream of the coding regions. Samples with ABO antigen typing and reverse serum typing discrepancies were screened in a total of 21 patients. All samples were subjected to serological testing and preliminary ABO genotyping (polymerase chain reaction with sequence-specific primers [PCR-SSP]), followed by single-molecule real-time (SMRT) sequencing to obtain complete ABO gene sequences. PCR sequence-based typing (PCR-SBT) was performed to validate the results. Of the 21 samples, 15 had common ABO types, and 6 had rare ABO subtypes. One new allele, ABO*B.NEW (c.861C>T), and one allelic base recombination event was identified. Forty-two haplotype sequences were obtained via SMRT sequencing with intronic single-nucleotide variants (SNVs) specific to the ABO allele, and all of the exon region sequences were consistent with the PCR-SBT results. SMRT sequencing is capable of accurately obtaining complete ABO gene sequences, distinguishing haplotypes and identifying allelic recombination.

Detection of hemophilia A genetic variants using third-generation long-read sequencing

BackgroundHemophilia A (HA) is an X-linked recessive genetic disorder caused by pathogenic variations of the factor VIII −encoding gene, F8 gene. Due to the large size and diverse types of variations in the F8 gene, causative mutations in F8 cannot be simultaneously detected in one step by traditional molecular analysis, and genetic molecular diagnosis and prenatal screening of HA still face significant difficulties and challenges in clinical practice. Therefore, we aimed to develop and validate an efficient, accurate, and time-saving method for the genetic detection of HA. MethodsA comprehensive analysis of hemophilia A (CAHEA) method based on long-range PCR and long-read sequencing (LRS) was used to detect F8 gene mutations in 14 clinical HA samples. The LRS results were compared with those of the conventional methods to evaluate the accuracy and sensitivity of the proposed approach. ResultsThe CAHEA method successfully identified 14 F8 variants in all probands, including 3 small insertion deletions, 4 single nucleotide variants, and 7 intron 22 inversions in a “one-step” manner, of which 2 small deletions have not been reported previously. Moreover, this method provided an opportunity to analyze the mechanism of rearrangement and the pathogenicity of F8 variants. The LRS results were validated and found to be in 100% agreement with those obtained using the conventional method. ConclusionOur proposed LRS-based F8 gene detection method is an accurate and reproducible genetic screening and diagnostic method with significant clinical value. It provides efficient, comprehensive, and accurate genetic screening and diagnostic services for individuals at high risk of HA as well as for premarital and prenatal populations.

Influenza A defective viral genomes and non-infectious particles are increased by host PI3K inhibition via anti-cancer drug alpelisib.

RNA viruses produce abundant defective viral genomes during replication, setting the stage for interactions between viral genomes that alter the course of pathogenesis. Harnessing these interactions to develop antivirals has become a recent goal of intense research focus. Despite decades of research, the mechanisms that regulate the production and interactions of Influenza A defective viral genomes are still unclear. The role of the host is essentially unexplored; specifically, it remains unknown whether host metabolism can influence the formation of defective viral genomes and the particles that house them. To address this question, we manipulated host cell anabolic signaling activity and monitored the production of defective viral genomes and particles by A/H1N1 and A/H3N2 strains, using a combination of single-cell immunofluorescence quantification, third-generation long-read sequencing, and the cluster-forming assay, a method we developed to titer defective and fully-infectious particles simultaneously. Here we show that alpelisib (Piqray), a highly selective inhibitor of mammalian Class 1a phosphoinositide-3 kinase (PI3K) receptors, significantly changed the proportion of defective particles and viral genomes (specifically deletion-containing viral genomes) in a strain-specific manner, under conditions that minimize multiple cycles of replication. Alpelisib pre-treatment of cells led to an increase in defective particles in the A/H3N2 strain, while the A/H1N1 strain showed a decrease in total viral particles. In the same infections, we found that defective viral genomes of polymerase and antigenic segments increased in the A/H1N1 strain, while the total particles decreased suggesting defective interference. We also found that the average deletion size in polymerase complex viral genomes increased in both the A/H3N2 and A/H1N1 strains. The A/H1N1 strain, additionally showed a dose-dependent increase in total number of defective viral genomes. In sum, we provide evidence that host cell metabolism can increase the production of defective viral genomes and particles at an early stage of infection, shifting the makeup of the infection and potential interactions among virions. Given that Influenza A defective viral genomes can inhibit pathogenesis, our study presents a new line of investigation into metabolic states associated with less severe flu infection and the potential induction of these states with metabolic drugs.

Next-Generation Sequencing and Emerging Technologies.

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.

CLAW: An automated Snakemake workflow for the assembly of chloroplast genomes from long-read data.

Chloroplasts are photosynthetic organelles in algal and plant cells that contain their own genome. Chloroplast genomes are commonly used in evolutionary studies and taxonomic identification and are increasingly becoming a target for crop improvement studies. As DNA sequencing becomes more affordable, researchers are collecting vast swathes of high-quality whole-genome sequence data from laboratory and field settings alike. Whole tissue read libraries sequenced with the primary goal of understanding the nuclear genome will inadvertently contain many reads derived from the chloroplast genome. These whole-genome, whole-tissue read libraries can additionally be used to assemble chloroplast genomes with little to no extra cost. While several tools exist that make use of short-read second generation and third-generation long-read sequencing data for chloroplast genome assembly, these tools may have complex installation steps, inadequate error reporting, poor expandability, and/or lack scalability. Here, we present CLAW (Chloroplast Long-read Assembly Workflow), an easy to install, customise, and use Snakemake tool to assemble chloroplast genomes from chloroplast long-reads found in whole-genome read libraries (https://github.com/aaronphillips7493/CLAW). Using 19 publicly available reference chloroplast genome assemblies and long-read libraries from algal, monocot and eudicot species, we show that CLAW can rapidly produce chloroplast genome assemblies with high similarity to the reference assemblies. CLAW was designed such that users have complete control over parameterisation, allowing individuals to optimise CLAW to their specific use cases. We expect that CLAW will provide researchers (with varying levels of bioinformatics expertise) with an additional resource useful for contributing to the growing number of publicly available chloroplast genome assemblies.

Resolving Genotype-Phenotype Discrepancies of the Kidd Blood Group System Using Long-Read Nanopore Sequencing.

Due to substantial improvements in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, fail to explain serological phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK, encoded by SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry-based platform at the Blood Transfusion Service, Zurich. Discrepant results from standard serological typing and donor genotyping were confirmed using commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (~24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors with both serological and genotype data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotypes, respectively. Remarkably, the remaining three cases were associated with a yet unknown deletion of ~5 kb spanning exons 9-10 of the JK*01 allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single-nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variants such as hybrid genes, deletions and duplications.

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.

Gut microbiome profiling of neonates using Nanopore MinION and Illumina MiSeq sequencing.

This study aimed to evaluate the difference in gut microbiomes between preterm and term infants using third-generation long-read sequencing (Oxford Nanopore Technologies, ONT) compared with an established gold standard, Illumina (second-generation short-read sequencing). A total of 69 fecal samples from 51 term (T) and preterm (P) infants were collected at 7 and 28 days of life. Gut colonization profiling was performed by 16S rRNA gene sequencing using ONT. We used Illumina to validate and compare the patterns in 13 neonates. Using bioinformatic analysis, we identified features that differed between P and T. Both T1 and P1 microbiomes were dominated by Firmicutes (Staphylococcus and Enterococcus), whereas sequentially showed dominant transitions to Lactobacillus (p < 0.001) and Streptococcus in T2 (p = 0.001), and pathogenic bacteria (Klebsiella) in P2 (p = 0.001). The abundance of beneficial bacteria (Bifidobacterium and Lactobacillus) increased in T2 (p = 0.026 and p < 0.001, respectively). These assignments were correlated with the abundance at the species-level. Bacterial α-diversity increased in T (p = 0.005) but not in P (p = 0.156), and P2 showed distinct β-diversity clustering than T2 (p = 0.001). The ONT reliably identified pathogenic bacteria at the genus level, and taxonomic profiles were comparable to those identified by Illumina at the genus level. This study shows that ONT and Illumina are highly correlated. P and T had different microbiome profiles, and the α- and β-diversity varied. ONT sequencing has potential for pathogen detection in neonates in clinical settings.

Metabolome and transcriptome signatures shed light on the anti-obesity effect of Polygonatum sibiricum.

Obesity has become one of the major threats to human health across the globe. The rhizomes of Polygonatum sibiricum have shown promising anti-obesity effect. However, the metabolic and genetic basis mediating this beneficial effect are not fully resolved. It is well known that older rhizomes of P. sibiricum exert stronger pharmacological effects. Here, we performed high-resolution metabolome profiling of P. sibiricum rhizomes at different growth stages, and identified that three candidate anti-obesity metabolites, namely phloretin, linoleic acid and α-linolenic acid, accumulated more in adult rhizomes. To elucidate the genetic basis controlling the accumulation of these metabolites, we performed transcriptome profiling of rhizomes from juvenile and adult P. sibiricum. Through third-generation long-read sequencing, we built a high-quality transcript pool of P. sibiricum, and resolved the genetic pathways involved in the biosynthesis and metabolism of phloretin, linoleic acid and α-linolenic acid. Comparative transcriptome analysis revealed altered expression of the genetic pathways in adult rhizomes, which likely lead to higher accumulation of these candidate metabolites. Overall, we identified several metabolic and genetic signatures related to the anti-obesity effect of P. sibiricum. The metabolic and transcriptional datasets generated in this work could also facilitate future research on other beneficial effects of this medicinal plant.

Improved Genome Assembly Resource of the Plant Pathogen Fusarium avenaceum

Fusarium avenaceum is a generalist plant pathogen of concern due to its potential to produce mycotoxins on plant products. Previous research efforts have sequenced and assembled genomes of F. avenaceum. However, those works relied on limited next-generation sequencing technologies that resulted in fragmented and incomplete genome assemblies. To address this, we utilized high-depth third-generation long-read sequencing and several different genome assembly software to generate a new, highly contiguous genome of F. avenaceum. Moreover, we conducted a thorough annotation of the genome using a mix of long-read cDNA and short-read RNAseq data. Our genome was more contiguous than the current reference genome Fustri1, matched the estimated genome size and chromosome number, and contained a similar number of annotated genes to other F. avenaceum genome assemblies. Lastly, we conducted a secondary metabolite (SM) cluster analysis that identified 60 gene clusters associated with SM biosynthesis, five more than the reference F. avenaceum genome. In conclusion, our genome and associated annotation information will help advance research on plant-fungal interactions, food safety, and Fusarium spp. diversity. [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 .

Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants.

In the era of blood group genomics, reference collections of complete and fully resolved blood group gene alleles have gained high importance. For most blood groups, however, such collections are currently lacking, as resolving full-length gene sequences as haplotypes (ie, separated maternal/paternal origin) remains exceedingly difficult with both Sanger and short-read next-generation sequencing. Using the latest third-generation long-read sequencing, we generated a collection of fully resolved sequences for all 6 main ABO allele groups: ABO∗A1/A2/B/O.01.01/O.01.02/O.02. We selected 77 samples from an ABO genotype data set (n= 25 200) of serologically typed Swiss blood donors. The entire ABO gene was amplified in 2 overlapping long-range polymerase chain reactions (covering ∼23.6 kb) and sequenced by long-read Oxford Nanopore sequencing. For quality validation, 2 samples per ABO group were resequenced using Illumina and Pacific Biosciences technology. All 154 full-length ABO sequences were resolved as haplotypes. We observed novel, distinct sequence patterns for each ABO group. Most genetic diversity was found between, not within, ABO groups. Phylogenetic tree and haplotype network analyses highlighted distinct clades of each ABO group. Strikingly, our data uncovered 4 genetic variants putatively specific for ABO∗A1, for which direct diagnostic targets are currently lacking. We validated A1-diagnostic potential using whole-genome data (n= 4872) of a multiethnic cohort. Overall, our sequencing strategy proved powerful for producing high-quality ABO haplotypes and holds promise for generating similar collections for other blood groups. The publicly available collection of 154 haplotypes will serve as a valuable resource for molecular analyses of ABO, as well as studies about the function and evolutionary history of ABO.

Sipunculus nudus genome provides insights into evolution of spiralian phyla and development

IntroductionSipunculus nudus is the most known species in the genus Sipunculus, distributed in tropical and subtropical coastal waters.MethodsPacBio sequencing and Illumina sequencing were combined for whole-genome sequencing of S. nudus. LC-MS/MS analysis was performed for the metabolomics of S. nudus.ResultsHerein, we report a 1.75 Gb complete genome assembly with a contig N50 size of 450 kb of the S. nudus based on a strategy combining third-generation long-read sequencing and Illumina sequencing. A total of 80391 protein-coding genes are annotated in this genome. Furthermore, gene family evolution analysis shows that S. nudus belonged to Mollusca or was close to Mollusca, but distinct with Annelida. Transcriptome analysis indicates the involvement of complex developmental events in larve. KEGG pathway analysis of differentially expressed genes showed that these DEGs were mainly enriched in the pathway of amino acid metabolism, lipid metabolism and transport and catabolism. LC-MS/MS analysis shows that S. nudus was rich in a variety of nutritional and functional components, such as carnitine, free amino acids, unsaturated fatty acids, inosine and methionine sulfoxide. Combined transcriptome with LC-MS/MS analysis, the results showed that gene expression and metabolic level involved in the inosine, arginine and proline biosynthesis of S. nudus during different growth stages were significantly changed.DiscussionOur genome assembly provides an important genome resource and new insight into the relationships of Sipuncula to other spiralian phyla. Meanwhile, transcriptome and LC-MS/MS analysis reveal the systematic gene expression profiles and metabolite components of S. nudus during different growth stages, which provide new insight into the exploration and development of bioactive molecules of S. nudus.

Long-Read Sequencing in Blood Group Genetics

Background: The key advantages of latest third-generation long-read sequencing (TGS) technologies include the ability to resolve long haplotypes and to characterize genomic regions that are challenging to analyze with short-read sequencing. Recent advancements in TGS technologies have significantly improved accuracy, a crucial requirement for the transition from research to diagnostic applications. Summary: In the field of immunohematology, the adoption of TGS is still in its early stages and published applications are scarce. An undeniable utility of TGS in blood group genomics is the ability to resolve ambiguous genotype-phenotype blood group results. In particular, hybrid genes and other large structural variants, as commonly found in the RHD/CE and MNS blood group systems, cause such discrepant results that can hardly be resolved by conventional methods. Long-read sequencing also greatly aids to generate high-standard reference alleles, establish haplotype sequence databases, or could even serve for high-resolution genotyping of all blood groups in parallel. Additionally, TGS holds the potential to close important knowledge gaps in blood group transcriptomics and epigenetics. Key Messages: The aims of this review were to examine the prospects of TGS technologies within the field of immunohematology and to highlight practical applications. Furthermore, we present a comprehensive overview of the existing and emerging wet-laboratory strategies for data generation, as well as a summary on bioinformatic data analysis methods. Finally, we provide an outlook on anticipated advancements in the near future.

Long-read assembly of major histocompatibility complex and killer cell immunoglobulin-like receptor genome regions in cynomolgus macaque

BackgroundThe major histocompatibility complex (MHC) and the killer cell immunoglobulin-like receptors (KIR) are key regulators of immune responses. The cynomolgus macaque, an Old World monkey species, can be applied as an important preclinical model for studying human diseases, including coronavirus disease 2019 (COVID-19). Several MHC-KIR combinations have been associated with either a poor or good prognosis. Therefore, macaques with a well-characterized immunogenetic profile may improve drug evaluation and speed up vaccine development. At present, a complete overview of the MHC and KIR haplotype organizations in cynomolgus macaques is lacking, and characterization by conventional techniques is hampered by the extensive expansion of the macaque MHC-B region that complicates the discrimination between genes and alleles.MethodsWe assembled complete MHC and KIR genomic regions of cynomolgus macaque using third-generation long-read sequencing approach. We identified functional Mafa-B loci at the transcriptome level using locus-specific amplification in a cohort of 33 Vietnamese cynomolgus macaques.ResultsThis is the first physical mapping of complete MHC and KIR gene regions in a Vietnamese cynomolgus macaque. Furthermore, we identified four functional Mafa-B loci (B2, B3, B5, and B6) and showed that alleles of the Mafa-I*01, -B*056, -B*034, and -B*001 functional lineages, respectively, are highly frequent in the Vietnamese cynomolgus macaque population.ConclusionThe insights into the MHC and KIR haplotype organizations and the level of diversity may refine the selection of animals with specific genetic markers for future medical research.

A Novel Bead-Capture Nanopore Sequencing Method for Large Structural Rearrangement Detection in Cancer

Rapid, cost-effective genomic stratification of structural rearrangements in cancer is often of vital importance when determining treatment; however, existing diagnostic cytogenetic and molecular testing fails to deliver the required speed when deployed at scale. Next-generation sequencing-based methods are widely used, but these can lack sensitivity and require batching of samples to be cost-effective, with long turnaround times. Here we present a novel method for rearrangement detection from genomic DNA based on third-generation long-read sequencing that overcomes these time and cost issues. The utility of this approach for the genomic stratification of patients with acute myeloid leukemia is shown based on detection of four of the most prevalent structural rearrangements. The method not only determines the precise genomic breakpoint for each expected rearrangement but also discovers and validates novel translocations in one-third of the tested samples, 80% of which involve known oncogenes. This method may prove to be a powerful tool for the diagnosis, genomic stratification, and characterization of cancers.

QitanTech Nanopore Long-Read Sequencing Enables Rapid Resolution of Complete Genomes of Multi-Drug Resistant Pathogens.

Advancement of novel sequencing technologies facilitates modern life science and medicine unprecedentedly. Exploring complete genome sequences of bacteria by long-read sequencing technology is significant for microbial genomics research. However, third-generation long-read sequencing technologies are available with limited choices, which generate technological barrier to scientific research. Recently, a novel QitanTech nanopore long-read sequencing technology has emerged in China, but the potential application and performance were unexplored. Herein, we comprehensively evaluated the feasibility of the emerging sequencing technology in assembling complete genomes of MDR pathogens. The results showed that 500 Mbp QitanTech nanopore sequencing data could be generated within 8 h in one flow cell with the standard library preparation method. The mean read length, longest read length, and mean read-level accuracy of QitanTech sequencing data were 6,041 bp, 57,037 bp, and 81.50% (LAST)/81.40% (Minimap2), respectively. Two routine assembly strategies including long-read assembly and hybrid assembly enable the achievement of complete bacterial genomes. The accuracy of assembled draft bacterial genomes with QitanTech long-read data could be improved up to 99.9% dramatically by polishing using accurate short-read data. Furthermore, the assembled bacterial genomes cover accurate structures of complex resistance plasmids harboring critical resistance genes such as tet(X), tmexCD-toprJ, and blaVIM–2, even the complex fusion MDR plasmid generated from homologous recombination. In conclusion, QitanTech nanopore sequencing, as a nanopore long-read sequencing technology launched in China, could be a good option for investigation of complex bacterial genomes. More potential applications based on this novel platform warrant investigations.

RRNA Analysis Based on Long-Read High-Throughput Sequencing Reveals a More Accurate Diagnostic for the Bacterial Infection of Ascites.

Traditional pathogenic diagnosis presents defects such as a low positivity rate, inability to identify uncultured microorganisms, and time-consuming nature. Clinical metagenomics next-generation sequencing can be used to detect any pathogen, compensating for the shortcomings of traditional pathogenic diagnosis. We report third-generation long-read sequencing results and second-generation short-read sequencing results for ascitic fluid from a patient with liver ascites and compared the two types of sequencing results with the results of traditional clinical microbial culture. The distribution of pathogenic microbial species revealed by the two types of sequencing results was quite different, and the third-generation sequencing results were consistent with the results of traditional microbial culture, which can effectively guide subsequent treatment. Short reads, the lack of amplification, and enrichment to amplify signals from trace pathogens, and host background noise may be the reasons for the high error in the second-generation short-read sequencing results. Therefore, we propose that long-read-based rRNA analysis technology is superior to the short-read shotgun-based metagenomics method in the identification of pathogenic bacteria.

Combined nanopore and single-molecule real-time sequencing survey of human betaherpesvirus 5 transcriptome

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

Amynthas corticis genome reveals molecular mechanisms behind global distribution

Earthworms (Annelida: Crassiclitellata) are widely distributed around the world due to their ancient origination as well as adaptation and invasion after introduction into new habitats over the past few centuries. Herein, we report a 1.2 Gb complete genome assembly of the earthworm Amynthas corticis based on a strategy combining third-generation long-read sequencing and Hi-C mapping. A total of 29,256 protein-coding genes are annotated in this genome. Analysis of resequencing data indicates that this earthworm is a triploid species. Furthermore, gene family evolution analysis shows that comprehensive expansion of gene families in the Amynthas corticis genome has produced more defensive functions compared with other species in Annelida. Quantitative proteomic iTRAQ analysis shows that expression of 147 proteins changed in the body of Amynthas corticis and 16 S rDNA sequencing shows that abundance of 28 microorganisms changed in the gut of Amynthas corticis when the earthworm was incubated with pathogenic Escherichia coli O157:H7. Our genome assembly provides abundant and valuable resources for the earthworm research community, serving as a first step toward uncovering the mysteries of this species, and may provide molecular level indicators of its powerful defensive functions, adaptation to complex environments and invasion ability.

Annotation of Full-Length Long Noncoding RNAs with Capture Long-Read Sequencing (CLS).

Metazoan genomes produce thousands of long-noncoding RNAs (lncRNAs), of which just a small fraction have been well characterized. Understanding their biological functions requires accurate annotations, or maps of the precise location and structure of genes and transcripts in the genome. Current lncRNA annotations are limited by compromises between quality and size, with many gene models being fragmentary or uncatalogued. To overcome this, the GENCODE consortium has developed RNA capture long-read sequencing (CLS), an approach combining targeted RNA capture with third-generation long-read sequencing. CLS provides accurate annotations at high-throughput rates. It eliminates the need for noisy transcriptome assembly from short reads, and requires minimal manual curation. The full-length transcript models produced are of quality comparable to present-day manually curated annotations. Here we describe a detailed CLS protocol, from probe design through long-read sequencing to creation of final annotations.