Circular Consensus Sequencing Research Articles

Long-read genome sequencing resolves complex genomic rearrangements in rare genetic syndromes

Long-read sequencing can often overcome the deficiencies in routine microarray or short-read technologies in detecting complex genomic rearrangements. Here we used Pacific Biosciences circular consensus sequencing to resolve complex rearrangements in two patients with rare genetic anomalies. Copy number variants (CNVs) identified by clinical microarray —chr8p deletion and chr8q duplication in patient 1, and interstitial deletions of chr18q in patient 2—were suggestive of underlying rearrangements. Long-read genome sequencing not only confirmed these CNVs but also revealed their genomic structures. In patient 1, we resolved a novel recombinant chromosome 8 (Rec8)-like rearrangement with a 3.43 Mb chr8q terminal duplication that was linked to a 7.25–8.21 Mb chr8p terminal deletion. In patient 2, we uncovered a novel complex rearrangement involving a 1.17 Mb rearranged segment and four interstitial deletions ranging from 9 bp to 12.39 Mb. Our results underscore the diversity of clinically relevant structural rearrangements and the power of long-read sequencing in unraveling their nuanced architectures.

The whole chromosome-level genome provides resources and insights into the endangered fish Percocypris pingi evolution and conservation

BackgroundPercocypris pingi (Tchang) was classified as Endangered on the Red List of China′s Vertebrates in 2015 and is widely distributed in the Upper Yangtze River. Although breeding and release into wild habitats have been performed for this commercially important fish in recent years, low genetic diversity has been found in wild populations. Genomic resources are strongly recommended before formulating and carrying out conservation strategies for P. pingi. Thus, there is an urgent need to conserve germplasm resources and improve the population diversity of P. pingi. To date, the whole genome of P. pingi has not been reported.ResultsIn our study, we constructed the first chromosome-level genome of P. pingi by high-throughput chromosome conformation capture (Hi-C) technology and PacBio long-read sequencing. The assembled genome was 1.7 Gb in size, with an N50 of 17,692 bp and a GC content from circular consensus sequencing of 37.67%. The Hi-C results again demonstrated that P. pingi was tetraploid (n = 98), with the genome consisting of 24-type and 25-type chromosomes. Chr.19 of the 24-type chromosomes in P. pingi resulted from the fusion of chr.19 and chr.22 in zebrafish. The divergence times between 24-type and 25-type chromosomes was around 6.1 million years ago. A total of 25,198 and 25,291 protein-coding genes were obtained from the 24-type and 25-type chromosomes, respectively. The ploidy of P. pingi is an allotetraploid. A total of 8,741 genes of P. pingi were clustered into 4,378 gene families that were shared with 14 other species, and the P. pingi genome had 68 unique gene families. Phylogenetic analyses indicated that P. pingi was most closely related to Schizothorax oconnori, and the genes were clustered on one branch. We identified 166 significantly expanded gene families and 173 significantly contracted gene families in P. pingi. The most enriched positive protein-coding genes, such as Bmp-4, Etfdh, homeobox protein HB9, and ATG3, were screened.ConclusionOur study provides a high-quality chromosome-anchored reference genome for P. pingi and provides sufficient information on the chromosomes, which will lead to valuable resources for genetic, genomic, and biological studies of P. pingi and for improving the genetic diversity, population size, and scientific conservation of endangered fish and other key cyprinid species in aquaculture.

Comparison of short-read and long-read next-generation sequencing technologies for determining HIV-1 drug resistance.

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.

Chromosome-level genome provides new insight into the overwintering process of Korla pear (Pyrus sinkiangensis Yu)

Korla pear has a unique taste and aroma and is a breeding parent of numerous pear varieties. It is susceptible to Valsa mali var. pyri, which invades bark wounded by freezing injury. Its genetic relationships have not been fully defined and could offer insight into the mechanism for freezing tolerance and disease resistance. We generated a high-quality, chromosome-level genome assembly for Korla pear via the Illumina and PacBio circular consensus sequencing (CCS) platforms and high-throughput chromosome conformation capture (Hi-C). The Korla pear genome is ~ 496.63 Mb, and 99.18% of it is assembled to 17 chromosomes. Collinearity and phylogenetic analyses indicated that Korla might be derived from Pyrus pyrifolia and that it diverged ~ 3.9-4.6 Mya. During domestication, seven late embryogenesis abundant (LEA), two dehydrin (DHN), and 54 disease resistance genes were lost from Korla pear compared with P. betulifolia. Moreover, 21 LEA and 31 disease resistance genes were common to the Korla pear and P. betulifolia genomes but were upregulated under overwintering only in P. betulifolia because key cis elements were missing in Korla pear. Gene deletion and downregulation during domestication reduced freezing tolerance and disease resistance in Korla pear. These results could facilitate the breeding of novel pear varieties with high biotic and abiotic stress resistance.

Chromosome-scale genome assembly and annotation of the tetraploid potato cultivar Diacol Capiro adapted to the Andean region.

Potato (Solanum tuberosum) is an essential crop for food security and is ranked as the third most important crop worldwide for human consumption. The Diacol Capiro cultivar holds the dominant position in Colombian cultivation, primarily catering to the food processing industry. This highly heterozygous, autotetraploid cultivar belongs to the Andigenum group and it stands out for its adaptation to a wide variety of environments spanning altitudes from 1,800 to 3,200 meters above sea level. Here, a chromosome-scale assembly, referred to as DC, is presented for this cultivar. The assembly was generated by combining circular consensus sequencing with proximity ligation Hi-C for the scaffolding and represents 2.369 Gb with 48 pseudochromosomes covering 2,091 Gb and an anchor rate of 88.26%. The reference genome metrics, including an N50 of 50.5 Mb, a BUSCO (Benchmarking Universal Single-Copy Orthologue) score of 99.38%, and an Long Terminal Repeat Assembly Index score of 13.53, collectively signal the achieved high assembly quality. A comprehensive annotation yielded a total of 154,114 genes, and the associated BUSCO score of 95.78% for the annotated sequences attests to their completeness. The number of predicted NLR (Nucleotide-Binding and Leucine-Rich-Repeat genes) was 2107 with a large representation of NBARC (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4) containing domains (99.85%). Further comparative analysis of the proposed annotation-based assembly with high-quality known potato genomes, showed a similar genome metrics with differences in total gene numbers related to the ploidy status. The genome assembly and annotation of DC presented in this study represent a valuable asset for comprehending potato genetics. This resource aids in targeted breeding initiatives and contributes to the creation of enhanced, resilient, and more productive potato varieties, particularly beneficial for countries in Latin America.

Comprehensive and deep evaluation of structural variation detection pipelines with third-generation sequencing data

BackgroundStructural variation (SV) detection methods using third-generation sequencing data are widely employed, yet accurately detecting SVs remains challenging. Different methods often yield inconsistent results for certain SV types, complicating tool selection and revealing biases in detection.ResultsThis study comprehensively evaluates 53 SV detection pipelines using simulated and real data from PacBio (CLR: Continuous Long Read, CCS: Circular Consensus Sequencing) and Nanopore (ONT) platforms. We assess their performance in detecting various sizes and types of SVs, breakpoint biases, and genotyping accuracy with various sequencing depths. Notably, pipelines such as Minimap2-cuteSV2, NGMLR-SVIM, PBMM2-pbsv, Winnowmap-Sniffles2, and Winnowmap-SVision exhibit comparatively higher recall and precision. Our findings also show that combining multiple pipelines with the same aligner, like pbmm2 or winnowmap, can significantly enhance performance. The individual pipelines’ detailed ranking and performance metrics can be viewed in a dynamic table: http://pmglab.top/SVPipelinesRanking.ConclusionsThis study comprehensively characterizes the strengths and weaknesses of numerous pipelines, providing valuable insights that can improve SV detection in third-generation sequencing data and inform SV annotation and function prediction.

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes.

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operon clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing strategies (PacBio circular consensus sequencing and two Nanopore approaches using unique molecular identifiers) and three tools for resolving amplicons sequence variants (ASVs) (USEARCH, VSEARCH, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essentially error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples, which enables us to identify previously undescribed microeukaryotic lineages.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang etal. 1993.

Long-read sequencing identifies an SVA_D retrotransposon insertion deep within the intron of ATP7A as a novel cause of occipital horn syndrome

BackgroundSINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a ‘copy-and-paste’ manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons...

NmTHC: a hybrid error correction method based on a generative neural machine translation model with transfer learning

BackgroundsThe single-pass long reads generated by third-generation sequencing technology exhibit a higher error rate. However, the circular consensus sequencing (CCS) produces shorter reads. Thus, it is effective to manage the error rate of long reads algorithmically with the help of the homologous high-precision and low-cost short reads from the Next Generation Sequencing (NGS) technology.MethodsIn this work, a hybrid error correction method (NmTHC) based on a generative neural machine translation model is proposed to automatically capture discrepancies within the aligned regions of long reads and short reads, as well as the contextual relationships within the long reads themselves for error correction. Akin to natural language sequences, the long read can be regarded as a special “genetic language” and be processed with the idea of generative neural networks. The algorithm builds a sequence-to-sequence(seq2seq) framework with Recurrent Neural Network (RNN) as the core layer. The before and post-corrected long reads are regarded as the sentences in the source and target language of translation, and the alignment information of long reads with short reads is used to create the special corpus for training. The well-trained model can be used to predict the corrected long read.ResultsNmTHC outperforms the latest mainstream hybrid error correction methods on real-world datasets from two mainstream platforms, including PacBio and Nanopore. Our experimental evaluation results demonstrate that NmTHC can align more bases with the reference genome without any segmenting in the six benchmark datasets, proving that it enhances alignment identity without sacrificing any length advantages of long reads.ConclusionConsequently, NmTHC reasonably adopts the generative Neural Machine Translation (NMT) model to transform hybrid error correction tasks into machine translation problems and provides a novel perspective for solving long-read error correction problems with the ideas of Natural Language Processing (NLP). More remarkably, the proposed methodology is sequencing-technology-independent and can produce more precise reads.

Full-length PacBio Amplicon Sequencing to Unveil RNA Editing Sites

Background: RNA editing enriches post-transcriptional sequence changes. Currently detecting RNA editing sites is mostly based on the Sanger sequencing platform and second-generation sequencing. However, detection with Sanger sequencing is limited by the disturbing background peaks using the direct sequencing method and the clone number using the clone sequencing method, while second-generation sequencing detection is constrained by its short read. Objective: We aimed to design a pipeline that can accurately detect RNA editing sites for full-length long-read amplicons to meet the requirement when focusing on a few specific genes of interest. Method: We developed a novel high-throughput RNA editing sites detection pipeline based on the PacBio circular consensus sequences sequencing which is accurate with high-throughput and long-read coverage. We tested the pipeline on cytosolic malate dehydrogenase in the hard-shelled mussel Mytilus coruscus and further validated it using direct Sanger sequencing. Results: Data generated from the PacBio circular consensus sequences (CCS) amplicons in three mussels were first filtered by quality and then selected by open reading frame. After filtering, 225-2047 sequences of the three mussels, respectively, were used to identify RNA editing sites. With corresponding genomic DNA sequences, we extracted 227-799 candidate RNA editing sites excluding heterozygous sites. We further figured out 7-11 final RESs using a new error model specially designed for RNA editing site detection. The resulting RNA editing sites all agree with the validation using the Sanger sequencing. Conclusion: We report a near-zero error rate method in identifying RNA editing sites of long-read amplicons with the use of PacBio CCS sequencing.

Semiconservative transmission of DNA N 6-adenine methylation in a unicellular eukaryote.

Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

DEGAP: Dynamic elongation of a genome assembly path.

Genome assembly remains to be a major task in genomic research. Despite the development over the past decades of different assembly software programs and algorithms, it is still a great challenge to assemble a complete genome without any gaps. With the latest DNA circular consensus sequencing (CCS) technology, several assembly programs can now build a genome from raw sequencing data to contigs; however, some complex sequence regions remain as unresolved gaps. Here, we present a novel gap-filling software, DEGAP (Dynamic Elongation of a Genome Assembly Path), that resolves gap regions by utilizing the dual advantages of accuracy and length of high-fidelity (HiFi) reads. DEGAP identifies differences between reads and provides 'GapFiller' or 'CtgLinker' modes to eliminate or shorten gaps in genomes. DEGAP adopts an iterative elongation strategy that automatically and dynamically adjusts parameters according to three complexity factors affecting the genome to determine the optimal extension path. DEGAP has already been successfully applied to decipher complex genomic regions in several projects and may be widely employed to generate more gap-free genomes.

Abstract 1763: Long-read sequencing of pediatric leukemia identifies clinically relevant genomic rearrangements

Abstract Clinical detection of genomic rearrangements currently depends on a range of complementary and overlapping methods including fluorescence in situ hybridization (FISH), karyotyping, and DNA and/or RNA sequencing. This requirement for multiple tests results in higher total costs, extended analysis time, and increased usage of sometimes limited tumor material. In our laboratory, we apply long high-fidelity reads to human samples using PacBio circular consensus sequencing (HiFi-GS) of 12-18kb DNA fragments, which offers better structural variant detection relative to short-read sequencing. Here, we assess the ability of HiFi-GS to detect clinically relevant genomic rearrangements (CRGRs) in pediatric leukemia. We selected 11 pediatric leukemia cases with corresponding normal (remission) samples, including 7 cases of B-cell acute lymphoblastic leukemia (B-ALL) and 4 cases of acute myeloid leukemia (AML); 5 of these 11 leukemia cases had known CRGRs as below. The remaining 6 leukemia cases had also undergone clinical genetic profiling via karyotyping, FISH, microarray, and sequencing yet a clinical grade genomic driver was not identified. DNA from each sample was sequenced on a Revio instrument (PacBio, Menlo Park, CA) to a target depth of 30x. The PacBio Human Whole Genome Sequencing (WGS) workflow was used to process HiFi reads for haplotagged alignment and phasing, followed by somatic structural variant calling using Severus. Subsequent analysis focused on the presence of break ends in known cancer-relevant genes. We found that HiFi-GS detected the known CRGRs in all 5 cases with prior findings, providing precise break ends and clarifying sometimes unclear cytogenetic observations (such as exact partner genes), in these cases. These included detection of an ETV6-RUNX1 fusion, NUP98-NSD1 fusion, KMT2A-AFF1 fusion, KMT2A-AFDN fusion, and RUNX1-RUNX1T1 fusion. HiFi sequencing also detected CRGRs in 2 of the 6 “undiagnosed cases”; namely, one case of ZNF384-TCF3 fusion and one case of KMT2A-MLLT10 fusion. The former can be cytogenetically cryptic, while the latter can often be missed due to its potentially complex nature. Our results demonstrate that HiFi-GS can reliably detect CRGRs, bringing us closer to the promise of a single comprehensive genetic test for cancer characterization. Additional work examining other somatic structural and copy number variants (i.e., inversions, deletions, and duplications) in these cases, as well as oncogenic sequence variants, is ongoing. Citation Format: Byunggil Yoo, Ayse Keskus, Chengpeng Bi, Lisa Lansdon, Tanveer Ahmad, Irina Pushel, Adam Walter, Margaret Gibson, Erin Guest, Tomi Pastinen, Mikhail Kolmogorov, Midhat S. Farooqi. Long-read sequencing of pediatric leukemia identifies clinically relevant genomic rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1763.

Enhancing transcriptome analysis in medicinal plants: multiple unigene sets in Astragalus membranaceus.

Astragalus membranaceus is a medicinal plant mainly used in East Asia and contains abundant secondary metabolites. Despite the importance of this plant, the available genomic and genetic information is still limited. De novo transcriptome construction is recognized as an essential method for transcriptome research when reference genome information is incomplete. In this study, we constructed three individual transcriptome sets (unigene sets) for detailed analysis of the phenylpropanoid biosynthesis pathway, a major metabolite of A. membranaceus. Set-1 was a circular consensus sequence (CCS) generated using PacBio sequencing (PacBio-seq). Set-2 consisted of hybridized assembled unigenes with Illumina sequencing (Illumina-seq) reads and PacBio CCS using rnaSPAdes. Set-3 unigenes were assembled from Illumina-seq reads using the Trinity software. Construction of multiple unigene sets provides several advantages for transcriptome analysis. First, it provides an appropriate expression filtering threshold for assembly-based unigenes: a threshold transcripts per million (TPM) ≥ 5 removed more than 88% of assembly-based unigenes, which were mostly short and low-expressing unigenes. Second, assembly-based unigenes compensated for the incomplete length of PacBio CCSs: the ends of the 5`/3` untranslated regions of phenylpropanoid-related unigenes derived from set-1 were incomplete, which suggests that PacBio CCSs are unlikely to be full-length transcripts. Third, more isoform unigenes could be obtained from multiple unigene sets; isoform unigenes missing in Set-1 were detected in set-2 and set-3. Finally, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that phenylpropanoid biosynthesis and carbohydrate metabolism were highly activated in A. membranaceus roots. Various sequencing technologies and assemblers have been developed for de novo transcriptome analysis. However, no technique is perfect for de novo transcriptome analysis, suggesting the need to construct multiple unigene sets. This method enables efficient transcript filtering and detection of longer and more diverse transcripts.

Endosymbiotic Fungal Diversity and Dynamics of the Brown Planthopper across Developmental Stages, Tissues, and Sexes Revealed Using Circular Consensus Sequencing.

Endosymbiotic fungi play an important role in the growth and development of insects. Understanding the endosymbiont communities hosted by the brown planthopper (BPH; Nilaparvata lugens Stål), the most destructive pest in rice, is a prerequisite for controlling BPH rice infestations. However, the endosymbiont diversity and dynamics of the BPH remain poorly studied. Here, we used circular consensus sequencing (CCS) to obtain 87,131 OTUs (operational taxonomic units), which annotated 730 species of endosymbiotic fungi in the various developmental stages and tissues. We found that three yeast-like symbionts (YLSs), Polycephalomyces prolificus, Ophiocordyceps heteropoda, and Hirsutella proturicola, were dominant in almost all samples, which was especially pronounced in instar nymphs 4-5, female adults, and the fat bodies of female and male adult BPH. Interestingly, honeydew as the only in vitro sample had a unique community structure. Various diversity indices might indicate the different activity of endosymbionts in these stages and tissues. The biomarkers analyzed using LEfSe suggested some special functions of samples at different developmental stages of growth and the active functions of specific tissues in different sexes. Finally, we found that the incidence of occurrence of three species of Malassezia and Fusarium sp. was higher in males than in females in all comparison groups. In summary, our study provides a comprehensive survey of symbiotic fungi in the BPH, which complements the previous research on YLSs. These results offer new theoretical insights and practical implications for novel pest management strategies to understand the BPH-microbe symbiosis and devise effective pest control strategies.

High-molecular-weight DNA extraction for broadleaved and conifer tree species

Abstract Plant genome sequencing based on long reads has increasingly been applied also to tree species in recent years. A crucial step in these genome projects is the successful extraction of high-molecular-weight DNA in high quality and sufficient quantity, which is imperative for long-read sequencing. The extraction of high-molecular-weight DNA in trees has been limited and difficult to conduct. To achieve successful extraction, modification of the protocol for tree species is necessary. Here, we present a protocol for high-molecular-weight DNA extraction from the broadleaved tree species Fraxinus excelsior and from the conifer species Taxus baccata. The protocol is based on nuclei isolation and is divided into two main steps, i.e. nuclei separation based on the nuclei isolation buffer and DNA extraction using the Nanobind® plant nuclei kit by Pacific Biosciences (PacBio). The protocol can be applied to different tree species to obtain high-molecular-weight DNA in high quality, which can be used for Oxford Nanopore Technologies (ONT) and PacBio sequencing. ONT sequencing using four high-molecular-weight DNA preparations from Fraxinus excel-sior resulted in different read length N50 values (12.91-38.19 kb) and total base output (5.81-23.17 Gb), emphasizing the complex nature of the sequencing pipeline of high-molecular-weight DNA. PacBio HiFi sequencing of Taxus baccata produced circular consensus sequencing reads with an average length of 12.9 kb and an N50 of 13.59 kb. Altogether, this study presents a protocol to obtain high-molecular-weight DNA from different challenging tree species and discusses several important points that can be considered when adapting the protocol to additional species.

Full-Length Transcriptome Sequencing and Identification of Genes Related to Terpenoid Biosynthesis in Cinnamomum migao H. W. Li

Cinnamomum migao H. W. Li is an evergreen woody plant that is only distributed in southwest China. The volatile oils from the fruits of C. migao have long been used as a special authentic medicinal herb by local ethnic minorities. Due to its low seed germination rate, destructive lumbering and low rates of artificial planting, C. migao is facing the danger of extinction. Therefore, it is urgent to exploit and protect this species using molecular biological technology, especially to target the genes involved in the biosynthesis of terpenoids in the volatile oil. However, the genomic data for this plant are not available. In this study, the transcriptome sequencing of C. migao was performed to obtain the key genes involved in terpenoid biosynthesis via a combination of full-length transcriptome and next-generation sequencing (NGS). More than 39.9 Gb of raw data was obtained and 515,929 circular consensus sequences (CCSs) were extracted. After clustering similar 472,858 full-length non-chimeric (FLNC) reads and correction with NGS data, 139,871 consensus isoforms were obtained. Meanwhile, 73,575 non-redundant transcripts were generated by removing redundant transcripts. Then, 70,427 isoforms were successfully annotated using public databases. Moreover, differentially expressed transcripts (DETs) in four different developmental stages of the C. migao fruit were analyzed and 5764 transcripts showed period-specific expression. Finally, 15 transcripts, 6 transcripts, and only 1 transcript were identified as being involved in the biosynthesis of sesquiterpenoids, diterpenoids, and monoterpenoids, respectively. This study provides a basis for future research in the gene mining, genetic breeding, and metabolic engineering of C. migao.

Bacterial pathogens associated with the plastisphere of surgical face masks and their dispersion potential in the coastal marine environment

Huge numbers of face masks (FMs) were discharged into the ocean during the coronavirus pandemic. These polymer-based artificial surfaces can support the growth of specific bacterial assemblages, pathogens being of particular concern. However, the potential risks from FM-associated pathogens in the marine environment remain poorly understood. Here, FMs were deployed in coastal seawater for two months. PacBio circular consensus sequencing of the full-length 16S rRNA was used for pathogen identification, providing enhanced taxonomic resolution. Selective enrichment of putative pathogens (e.g., Ralstonia pickettii) was found on FMs, which provided a new niche for these pathogens rarely detected in the surrounding seawater or the stone controls. The total relative abundance of the putative pathogens in FMs was higher than in seawater but lower than in the stone controls. FM exposure during the two months resulted in 3% weight loss and the release of considerable amounts of microfibers. The ecological assembly process of the putative FM-associated pathogens was less impacted by the dispersal limitation, indicating that FM-derived microplastics can serve as vectors of most pathogens for their regional transport. Our results indicate a possible ecological risk of FMs for marine organisms or humans in the coastal and potentially in the open ocean.

A genomic platform for surveillance and antigen discovery in Plasmodium spp. using long-read amplicon sequencing

Many vaccine candidate proteins in the malaria parasite Plasmodium falciparum are under strong immunological pressure and confer antigenic diversity. We present a sequencing and data analysis platform for the genomic surveillance of the insertion or deletion (indel)-rich antigens merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP), and CSP from P.falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers per gene to asymmetrically barcode and identify multiclonal infections in pools of up to 384 samples. With msp2, we validated the method using 235 mock infections combining 10 synthetic variants at different concentrations and infection complexities. We applied this strategy to P.falciparum isolates from a longitudinal cohort in Tanzania. Finally, we constructed an analysis pipeline that streamlines the processing and interpretation of epidemiological and antigenic diversity data from demultiplexed FASTQ files. This platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.