Circular Consensus Sequencing Reads Research Articles

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.

DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing

Long single-molecular sequencing technologies, such as PacBio circular consensus sequencing (CCS) and nanopore sequencing, are advantageous in detecting DNA 5-methylcytosine in CpGs (5mCpGs), especially in repetitive genomic regions. However, existing methods for detecting 5mCpGs using PacBio CCS are less accurate and robust. Here, we present ccsmeth, a deep-learning method to detect DNA 5mCpGs using CCS reads. We sequence polymerase-chain-reaction treated and M.SssI-methyltransferase treated DNA of one human sample using PacBio CCS for training ccsmeth. Using long (≥10 Kb) CCS reads, ccsmeth achieves 0.90 accuracy and 0.97 Area Under the Curve on 5mCpG detection at single-molecule resolution. At the genome-wide site level, ccsmeth achieves >0.90 correlations with bisulfite sequencing and nanopore sequencing using only 10× reads. Furthermore, we develop a Nextflow pipeline, ccsmethphase, to detect haplotype-aware methylation using CCS reads, and then sequence a Chinese family trio to validate it. ccsmeth and ccsmethphase can be robust and accurate tools for detecting DNA 5-methylcytosines.

Chromosome-level assembly of Dictyophora rubrovolvata genome using third-generation DNA sequencing and Hi-C analysis.

Dictyophora rubrovolvata, a rare edible mushroom with both nutritional and medicinal values, was regarded as the "queen of the mushroom" for its attractive appearance. Dictyophora rubrovolvata has been widely cultivated in China in recent years, and many researchers were focusing on its nutrition, culture condition, and artificial cultivation. Due to a lack of genomic information, research on bioactive substances, cross breeding, lignocellulose degradation, and molecular biology is limited. In this study, we report a chromosome-level reference genome of D. rubrovolvata using the PacBio single-molecule real-time-sequencing technique and high-throughput chromosome conformation capture (Hi-C) technologies. A total of 1.83 Gb circular consensus sequencing reads representing ∼983.34 coverage of the D. rubrovolvata genome were generated. The final genome was assembled into 136 contigs with a total length of 32.89 Mb. The scaffold and contig N50 length were 2.71 and 2.48 Mb, respectively. After chromosome-level scaffolding, 11 chromosomes with a total length of 28.24 Mb were constructed. Genome annotation further revealed that 9.86% of the genome was composed of repetitive sequences, and a total of 508 noncoding RNA (rRNA: 329, tRNA: 150, ncRNA: 29) were annotated. In addition, 9,725 protein-coding genes were predicted, among which 8,830 (90.79%) genes were predicted using homology or RNA-seq. Benchmarking Universal Single-Copy Orthologs results further revealed that there were 80.34% complete single-copy fungal orthologs. In this study, a total of 360 genes were annotated as belonging to the carbohydrate-active enzymes family. Further analysis also predicted 425 cytochromes P450 genes, which can be classified into 41 families. This highly accurate, chromosome-level reference genome of D. rubrovolvata will provide essential genomic information for understanding the molecular mechanism in its fruiting body formation during morphological development and facilitate the exploitation of medicinal compounds produced by this mushroom.

Error analysis of the PacBio sequencing CCS reads.

Third generation sequencing technologies such as Pacific Biosciences and Oxford Nanopore provide faster, cost-effective and simpler assembly process generating longer reads than the ones in the next generation sequencing. However, the error rates of these long reads are higher than those of the short reads, resulting in an error correcting process before the assembly such as using the Circular Consensus Sequencing (CCS) reads in PacBio sequencing machines. In this paper, we propose a probabilistic model for the error occurrence along the CCS reads. We obtain the error probability of any arbitrary nucleotide as well as the base calling Phred quality score of the nucleotides along the CCS reads in terms of the number of sub-reads. Furthermore, we derive the error rate distribution of the reads in relation to the pass number. It follows the binomial distribution which can be approximated by the normal distribution for long reads. Finally, we evaluate our proposed model by comparing it with three real PacBio datasets, namely, Lambda, and E. coli genomes, and Alzheimer's disease targeted experiment.

Detection of hemoglobin H disease by long molecule sequencing.

BackgroundHemoglobin H (Hb H) disease is a moderate‐to‐severe form of α‐thalassemia (α‐thal), and parts of patients may require intermittent transfusion therapy, especially during intercurrent illness. However, rare Hb H diseases remain undetected using routine methods being outside of the testing scope. In this study, we present an approach to detecting Hb H disease by long molecule sequencing (LMS).MethodsA total of 206 known genotype samples were collected and carried to blind detected by LMS on the PacBio Sequel platform. Circular consensus sequencing reads were aligned to the hg19 reference genome using Free‐Bayes finished LMS. LMS accuracy would be compared with routine methods, including Gap‐PCR and PCR‐Reverse dot blot hybridization (PCR–RDB).ResultsThe assay could detect carriers of both deletion and point mutations. It had an overall accuracy of 100% when compared with routine methods. In addition, LMS detected six mutations based on routine methods and corrected three case results. Hb H diseases were identified using LMS, whether a common or rare genotype, a deletion or non‐deletion genotype. However, two cases of Hb H disease were misdiagnosed using routine methods.ConclusionsLong molecule sequencing can be suggested as a rapid and reliable assay to detect probable carriers of hemoglobinopathies. LMS accurately identified the common and rare genotypes of Hb H disease.

Comparison of ONT and CCS sequencing technologies on the polyploid genome of a medicinal plant showed that high error rate of ONT reads are not suitable for self-correction

BackgroundMany medicinal plants are known for their complex genomes with high ploidy, heterozygosity, and repetitive content which pose severe challenges for genome sequencing of those species. Long reads from Oxford nanopore sequencing technology (ONT) or Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing offer great advantages in de novo genome assembly, especially for complex genomes with high heterozygosity and repetitive content. Currently, multiple allotetraploid species have sequenced their genomes by long-read sequencing. However, we found that a considerable proportion of these genomes (7.9% on average, maximum 23.7%) could not be covered by NGS (Next Generation Sequencing) reads (uncovered region by NGS reads, UCR) suggesting the questionable and low-quality of those area or genomic areas that can’t be sequenced by NGS due to sequencing bias. The underlying causes of those UCR in the genome assembly and solutions to this problem have never been studied.MethodsIn the study, we sequenced the tetraploid genome of Veratrum dahuricum (Turcz.) O. Loes (VDL), a Chinese medicinal plant, with ONT platform and assembled the genome with three strategies in parallel. We compared the qualities, coverage, and heterozygosity of the three ONT assemblies with another released assembly of the same individual using reads from PacBio circular consensus sequencing (CCS) technology, to explore the cause of the UCR.ResultsBy mapping the NGS reads against the three ONT assemblies and the CCS assembly, we found that the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%). And alignment between ONT assemblies and CCS assembly showed that most UCR can be aligned with CCS assembly. So, we conclude that the UCRs in ONT assembly are low-quality sequences with a high error rate that can’t be aligned with short reads, rather than genomic regions that can’t be sequenced by NGS. Further comparison among the intermediate versions of ONT assemblies showed that the most probable origin of those errors is a combination of artificial errors introduced by “self-correction” and initial sequencing error in long reads. We also found that polishing the ONT assembly with CCS reads can correct those errors efficiently.ConclusionsThrough analyzing genome features and reads alignment, we have found the causes for the high proportion of UCR in ONT assembly of VDL are sequencing errors and additional errors introduced by self-correction. The high error rates of ONT-raw reads make them not suitable for self-correction prior to allotetraploid genome assembly, as the self-correction will introduce artificial errors to > 5% of the UCR sequences. We suggest high-precision CCS reads be used to polish the assembly to correct those errors effectively for polyploid genomes.

Integrated Full-Length Transcriptome and MicroRNA Sequencing Approaches Provide Insights Into Salt Tolerance in Mangrove (Sonneratia apetala Buch.-Ham.).

MicroRNAs (miRNAs) are small RNA molecules that serve as key players in plant stress responses. Although stress-regulated miRNAs have been explored in various plants, they are not well studied in mangroves. Herein, we combined PacBio isoform sequencing (Iso-Seq) with BGISEQ short-read RNA-seq to probe the role of miRNAs in the salt stress response of the mangrove plant, Sonneratia apetala Buch.-Ham. A total of 1,702,463 circular consensus sequencing reads were generated that produced 295,501 nonredundant full-length transcripts from the leaves of a 1-year-old S. apetala. After sequencing nine small RNA libraries constructed from control and 1- and 28-day 300 mM NaCl treatments, we identified 143 miRNAs (114 known and 29 novel) from a total of >261 million short reads. With the criteria of |log2FC| ≥ 1 and q-value < 0.05, 42 and 70 miRNAs were differentially accumulated after 1- and 28-day salt treatments, respectively. These differential accumulated miRNAs potentially targeted salt-responsive genes encoding transcription factors, ion homeostasis, osmotic protection, and detoxificant-related proteins, reminiscent of their responsibility for salinity adaptation in S. apetala. Particularly, 62 miRNAs were Sonneratia specific under salt stress, of which 34 were co-expressed with their 131 predicted targets, thus producing 140 miRNA–target interactions. Of these, 82 miRNA-target pairs exhibited negative correlations. Eighteen miRNA targets were categorized for the ‘environmental information processing’ during KEGG analysis and were related to plant hormone signal transduction (ko04075), MAPK signaling pathway–plant (ko04016), and ABC transporters (ko02010). These results underscored miRNAs as possible contributors to mangrove success in severe environments and offer insights into an miRNA-mediated regulatory mechanism of salt response in S. apetala.

QuasiSeq: profiling viral quasispecies via self-tuning spectral clustering with PacBio long sequencing reads.

The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads. We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills. QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542. Supplementary data are available at Bioinformatics online.

Chromosome-scale Cerasus humilis genome assembly reveals gene family evolution and possible genomic basis of calcium accumulation in fruits

Chinese dwarf cherry (Cerasus humilis), which is well known as “calcium fruit” for its high calcium content, is widely utilized in North China for its ecological and economical value. Understanding the evolutionary history and genetic basis underlying the calcium accumulation in this fruit is a fundamental goal in comparative genomics. In this study, we obtained a full phased chromosome-scale C. humilis genome for the cultivar “zhisha” using PacBio circular consensus sequencing reads and Phase genomics Proximo Hi-C technologies. The final assembled genome lengths were 235.41 and 237.35 Mb for primary assembly (p0) and alternate haplotig (p1), respectively, with an N50 contig length of 3.23 Mb. The genome contained 26,800 protein-coding genes, while 41.33% of the genome length consisted of TEs. Phylogenetic analysis revealed that C. humilis displayed a close relationship with Prunus and phylogenetically diverged from its common ancestor ∼23.2 million years ago (MYA). Functional enrichment of expanded gene families in C. humilis genome highlighted genes involved in signal transduction, antiporter activity, polygalacturonase activity, auxin efflux transmembrane transporter activity, and auxin efflux, which is related to calcium transport and distribution. Phylogenetic analysis coupled with tissue-specific expression patterns indicated that PSGs WAK2 and CBL10, may also be responsible for the high Ca2+accumulation ability in C. humilis. These results glean tremendous insight into the fundamental genetic basis of the high calcium content in C. humilis fruit. In addition, the reference C. humilis genome we present in this study will be valuable resources for molecular breeding and genetic research of this fruit species.

EST-SSR Marker Development and Full-Length Transcriptome Sequence Analysis of Tiger Lily (Lilium lancifolium Thunb).

The fast advancement and deployment of sequencing technologies after the Human Genome Project have greatly increased our knowledge of the eukaryotic genome sequences. However, due to technological concerns, high-quality genomic data has been confined to a few key organisms. Moreover, our understanding of which portions of genomes make up genes and which transcript isoforms synthesize these genes is scarce. Therefore, the current study has been designed to explore the reliability of the tiger lily (Lilium lancifolium Thunb) transcriptome. The PacBio-SMRT was used for attaining the complete transcriptomic profile. We obtained a total of 815,624 CCS (Circular Consensus Sequence) reads with an average length of 1295 bp. The tiger lily transcriptome has been sequenced for the first time using third-generation long-read technology. Furthermore, unigenes (38,707), lncRNAs (6852), and TF members (768) were determined based on the transcriptome data, followed by evaluating SSRs (3319). It has also been revealed that 105 out of 128 primer pairs effectively amplified PCR products. Around 15,608 transcripts were allocated to 25 distinct KOG Clusters, and 10,706 unigenes were grouped into 52 functional categories in the annotated transcripts. Until now, no tiger lily lncRNAs have been discovered. Results of this study may serve as an extensive set of reference transcripts and help us learn more about the transcriptomes of tiger lilies and pave the path for further research.

Characterization of the early gene expression profile in Populus ussuriensis under cold stress using PacBio SMRT sequencing integrated with RNA-seq reads.

Populus ussuriensis is an important and fast-growing afforestation plant species in north-eastern China. The whole-genome sequencing of P. ussuriensis has not been completed. Also, the transcriptional network of P. ussuriensis response to cold stress remains unknown. To unravel the early response of P. ussuriensis to chilling (3 °C) stress and freezing (-3 °C) stresses at the transcriptional level, we performed single-molecule real-time (SMRT) and Illumina RNA sequencing for P. ussuriensis. The SMRT long-read isoform sequencing led to the identification of 29,243,277 subreads and 575,481 circular consensus sequencing reads. Approximately 50,910 high-quality isoforms were generated, and 2272 simple sequence repeats and 8086 long non-coding RNAs were identified. The Ca2+ content and abscisic acid (ABA) content in P. ussuriensis were significantly increased under cold stresses, while the value in the freezing stress treatment group was significantly higher than the chilling stress treatment group. A total of 49 genes that are involved in the signal transduction pathways related to perception and transmission of cold stress signals, such as the Ca2+ signaling pathway, ABA signaling pathway and MAPK signaling cascade, were found to be differentially expressed. In addition, 158 transcription factors from 21 different families, such as MYB, WRKY and AP2/ERF, were differentially expressed during chilling and freezing treatments. Moreover, the measurement of physiological indicators and bioinformatics observations demonstrated the altered expression pattern of genes involved in reactive oxygen species balance and the sugar metabolism pathway during chilling and freezing stresses. This is the first report of the early responses of P. ussuriensis to cold stress, which lays the foundation for future studies on the regulatory mechanisms in cold-stress response. In addition the full-length reference transcriptome of P. ussuriensis deciphered could be used in future studies on P. ussuriensis.

SKSV: ultrafast structural variation detection from circular consensus sequencing reads.

Circular consensus sequencing reads are promising for the comprehensive detection of structural variants (SVs). However, alignment-based SV calling pipelines are computationally intensive due to the generation of complete read-alignments and its post-processing. Herein, we propose a SKeleton-based analysis toolkit for Structural Variation detection (SKSV). Benchmarks on real and simulated datasets demonstrate that SKSV has an order of magnitude of faster speed than state-of-the-art SV calling approaches; moreover, it achieves higher F1 scores for various types of SVs. SKSV is available from https://github.com/ydLiu-HIT/SKSV. Supplementary data are available at Bioinformatics online.

Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases.

To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10−4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10− 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases.

Robust Benchmark Structural Variant Calls of An Asian Using State-of-the-art Long-read Sequencing Technologies

The importance of structural variants (SVs) for human phenotypes and diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of these SV detection approaches, we established an Asian reference material by characterizing the genome of an Epstein-Barr virus (EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers, including 109× Pacific Biosciences (PacBio) continuous long reads (CLRs), 22× PacBio circular consensus sequencing (CCS) reads, 104× Oxford Nanopore Technologies (ONT) long reads, and 114× Bionano optical mapping platform, and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing, demonstrating the robustness of our SV calls. Combining trio-binning-based haplotype assemblies, we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions (CHCRs), which covered 1.46 gigabase pairs (Gb) and 6882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical research.

Sensitive alignment using paralogous sequence variants improves long-read mapping and variant calling in segmental duplications.

The ability to characterize repetitive regions of the human genome is limited by the read lengths of short-read sequencing technologies. Although long-read sequencing technologies such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies can potentially overcome this limitation, long segmental duplications with high sequence identity pose challenges for long-read mapping. We describe a probabilistic method, DuploMap, designed to improve the accuracy of long-read mapping in segmental duplications. It analyzes reads mapped to segmental duplications using existing long-read aligners and leverages paralogous sequence variants (PSVs)—sequence differences between paralogous sequences—to distinguish between multiple alignment locations. On simulated datasets, DuploMap increased the percentage of correctly mapped reads with high confidence for multiple long-read aligners including Minimap2 (74.3–90.6%) and BLASR (82.9–90.7%) while maintaining high precision. Across multiple whole-genome long-read datasets, DuploMap aligned an additional 8–21% of the reads in segmental duplications with high confidence relative to Minimap2. Using DuploMap-aligned PacBio circular consensus sequencing reads, an additional 8.9 Mb of DNA sequence was mappable, variant calling achieved a higher F1 score and 14 713 additional variants supported by linked-read data were identified. Finally, we demonstrate that a significant fraction of PSVs in segmental duplications overlaps with variants and adversely impacts short-read variant calling.

Comparative analysis of full-length transcriptomes based on hybrid population reveals regulatory mechanisms of anthocyanin biosynthesis in sweet potato (Ipomoea batatas (L.) Lam)

BackgroundSweet potato (Ipomoea batatas (L.) Lam.) is a highly heterozygous autohexaploid crop with high yield and high anthocyanin content. Purple sweet potato is the main source of anthocyanins, and the mechanism of anthocyanin biosynthesis in storage roots has not been fully revealed.ResultsIn order to reveal the mechanism of anthocyanin biosynthesis and identify new homologous genes involved in anthocyanin biosynthesis in the storage roots of sweet potato, we used Ningzishu 1 and Jizishu 2 as parents to construct a F1 hybrid population. Seven anthocyanin-containing lines and three anthocyanin-free lines were selected for full-length and second-generation transcriptome analyses. A total of 598,375 circular consensus sequencing reads were identified from full-length transcriptome sequencing. After analysis and correction of second-generation transcriptome data, 41,356 transcripts and 18,176 unigenes were obtained. Through a comparative analysis between anthocyanin-containing and anthocyanin-free groups 2329 unigenes were found to be significantly differentially expressed, of which 1235 were significantly up-regulated and 1094 were significantly down-regulated. GO enrichment analysis showed that the differentially expressed unigenes were significantly enriched in molecular function and biological process. KEGG enrichment analysis showed that the up-regulated unigenes were significantly enriched in the flavonoid biosynthesis and phenylpropanoid biosynthesis pathways, and the down-regulated unigenes were significantly enriched in the plant hormone signal transduction pathway. Weighted gene co-expression network analysis of differentially expressed unigenes revealed that anthocyanin biosynthesis genes were co-expressed with transcription factors such as MYB, bHLH and WRKY at the transcription level.ConclusionsOur study will shed light on the regulatory mechanism of anthocyanin biosynthesis in sweet potato storage roots at the transcriptome level.

Long-Molecule Sequencing: A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and β-Thalassemia Carriers

Multiple molecular tests are currently needed for accurate carrier testing for thalassemia. Therefore, long-molecule sequencing (LMS) was evaluated as an alternate on the PacBio Sequel platform for genotyping carriers of α-thalassemia or β-thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and β (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using FreeBayes software version 1.2.0. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common South East Asian, -α3.7, and -α4.2 deletions and four different rare single-nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the most common Chinese insertion and deletion variant c.126_129delCTTT and 14 different SNVs/insertions and deletions and could discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate, and cost-effective genotyping method. With further test coverage to additionally include detection of other clinically significant HBA1/2 copy number variations, such as the Thai, Mediterranean, and Filipino deletions, LMS may eventually serve as a comprehensive method for large-scale thalassemia carrier screening.

Highly accurate mtGenome haplotypes from long-read SMRT sequencing can distinguish between monozygotic twins

Discriminating between monozygotic twins (MZ) remains a challenge in the field of forensics globally. It is very difficult to find sequence variants within MZ twins, despite using ultra-deep next generation sequencing (NGS) for nuclear DNA. However, mitochondrial DNA might be a potential marker owing to its higher mutation rate and easier sequencing via NGS. Here, we aimed to introduce a long-read single molecule real-time sequencing (SMRT) strategy, with better continuity and fewer alignment errors, to obtain more accurate mitochondrial genome (mtGenome) sequence on the Sequel platform. Compared to Ion Torrent Personal Genome Machine (PGM), the long-read SMRT sequencing strategy generated highly accurate and mapped circular consensus sequence (CCS) reads and exhibited robust performance in terms of reliable repeatability, consistent coverage pattern, and balanced strands in mtGenome recovery. Moreover, the long-read SMRT strategy exhibited superior ability to not only identify accurate haplotypes but also discover a total of 785 low-level variants within 16 MZ twin pairs with threshold of 2% and 20 CCS reads with Q30 quality. Taken together, our findings suggested the long-read SMRT technology as an appreciable strategy for obtaining accurate mitotypes and providing a promising solution for distinguishing between MZ twin pairs in forensic genetics.

Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity.

High-throughput DNA metabarcoding of amplicon sizes below 500bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long-read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500-bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high-quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the ‘genome in a bottle’ (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.