Short-read Next-generation Sequencing Research Articles

Two Novel Variants in PI4KA in a Family Presenting With Hereditary Spastic Paraparesis: A Case Report.

To report novel biallelic PI4KA variants in a family presenting with pure hereditary spastic paraparesis. Two affected sisters presented with unsolved hereditary spastic paraparesis and underwent clinical and imaging assessments. This was followed by short-read next-generation sequencing. Analysis of next-generation sequencing data uncovered compound heterozygous variants in PI4KA (NM_058004.4: c.[3883C>A];[5785A>C]; p.[(His1295Asn);(Thr1929Pro)]. Using ACMG guidelines, both variants were classified as likely pathogenic. Here, next-generation sequencing revealed 2 novel compound heterozygous variants in the phosphatidylinositol 4-kinase alpha gene (PI4KA) in 2 sisters presenting with progressive pure hereditary spastic paraparesis. Pathogenic variants in PI4KA have previously been associated with a spectrum of disorders including autosomal recessive perisylvian polymicrogyria, with cerebellar hypoplasia, arthrogryposis, and pure spastic paraplegia. The cases presented in this study expand the phenotypic spectrum associated with PI4KA variants and contribute new likely pathogenic variants for testing in patients with otherwise unsolved hereditary spastic paraparesis.

Long-read next-generation sequencing for molecular diagnosis of pediatric endocrine disorders.

Recent advances in long-read next-generation sequencing (NGS) have enabled researchers to identify several pathogenic variants overlooked by short-read NGS, array-based comparative genomic hybridization, and other conventional methods. Long-read NGS is particularly useful in the detection of structural variants and repeat expansions. Furthermore, it can be used for mutation screening in difficultto- sequence regions, as well as for DNA-methylation analyses and haplotype phasing. This mini-review introduces the usefulness of long-read NGS in the molecular diagnosis of pediatric endocrine disorders.

Combined approaches, including long-read sequencing, address the diagnostic challenge of HYDIN in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD), a disorder of the motile cilia, is now recognised as an underdiagnosed cause of bronchiectasis. Accurate PCD diagnosis comprises clinical assessment, analysis of cilia and the identification of biallelic variants in one of 50 known PCD-related genes, including HYDIN. HYDIN-related PCD is underdiagnosed due to the presence of a pseudogene, HYDIN2, with 98% sequence homology to HYDIN. This presents a significant challenge for Short-Read Next Generation Sequencing (SR-NGS) and analysis, and many diagnostic PCD gene panels do not include HYDIN. We have used a combined approach of SR-NGS with bioinformatic masking of HYDIN2, and state-of-the-art long-read Nanopore sequencing (LR_NGS), together with analysis of respiratory cilia including transmission electron microscopy and immunofluorescence to address the underdiagnosis of HYDIN as a cause of PCD. Bioinformatic masking of HYDIN2 after SR-NGS facilitated the detection of biallelic HYDIN variants in 15 of 437 families, but compromised the detection of copy number variants. Supplementing testing with LR-NGS detected HYDIN deletions in 2 families, where SR-NGS had detected a single heterozygous HYDIN variant. LR-NGS was also able to confirm true homozygosity in 2 families when parental testing was not possible. Utilising a combined genomic diagnostic approach, biallelic HYDIN variants were detected in 17 families from 242 genetically confirmed PCD cases, comprising 7% of our PCD cohort. This represents the largest reported HYDIN cohort to date and highlights previous underdiagnosis of HYDIN-associated PCD. Moreover this provides further evidence for the utility of LR-NGS in diagnostic testing, particularly for regions of high genomic complexity.

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

In the practice of clinical genetics, gene testing is usually guided by clinical diagnosis. When dealing with rare diseases, it is often necessary to create new test systems. The handling of a gene with a substantial number of exons poses a challenge both in sequential Sanger sequencing for each exon, and in the setup of capture probes to each exon for next-generation sequencing (NGS). We present very long amplicon sequencing (vLAS), an optimized long-range polymerase chain reaction (PCR)-based NGS method that overcomes this challenge. By utilizing approximately 20 Kb long PCR products and short-read NGS, vLAS is emerging as a highly adaptable and effective solution, especially for genes with numerous exons concentrated in a limited genomic region. Here, we demonstrate vLAS in the analysis of five patients with type I and two with type II collagenopathies. The integration of user-friendly NGS methods into genetic diagnosis enhances the practicality of clinical genetics.

Next generation sequencing (NGS)-based approach to diagnosing Algerian patients with suspected inborn errors of immunity (IEIs)

The advent of next-generation sequencing (NGS) technologies has greatly expanded our understanding of both the clinical spectra and genetic landscape of inborn errors of immunity (IEIs). Endogamous populations may be enriched for unique, ancestry-specific disease-causing variants, a consideration that significantly impacts molecular testing and analysis strategies. Herein, we report on the application of a 2-step NGS-based testing approach beginning with targeted gene panels (TGPs) tailored to specific IEI subtypes and reflexing to whole exome sequencing (WES) if negative for Northwest Algerian patients with suspected IEIs. Our overall diagnostic yield of 57% is comparable to others broadly applying short-read NGS to IEI detection, but data from our localized cohort show some similarities and differences from NGS studies performed on larger regional IEI cohorts. This suggests the importance of tailoring diagnostic strategies to local demographics and needs, but also highlights ongoing concerns inherent to the application of genomics for clinical IEI diagnostics.

Closing the Gap - Detection of 5q-Spinal Muscular Atrophy by Short-Read Next-Generation Sequencing and Unexpected Results in a Diagnostic Patient Cohort.

The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV). 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients). Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.

Nanopore long-read next-generation sequencing for detection of mitochondrial DNA large-scale deletions.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.

Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms

The exploration of oral microbiome has been increasing due to its relatedness with various systemic diseases, but standardization of saliva sampling for microbiome analysis has not been established, contributing to the lack of data comparability. Here, we evaluated the factors that influence the microbiome data. Saliva samples were collected by the two collection methods (passive drooling and mouthwash) using three saliva-preservation methods (OMNIgene, DNA/RNA shield, and simple collection). A total of 18 samples were sequenced by both Illumina short-read and Nanopore long-read next-generation sequencing (NGS). The component of the oral microbiome in each sample was compared with alpha and beta diversity and the taxonomic abundances, to find out the effects of factors on oral microbiome data. The alpha diversity indices of the mouthwash sample were significantly higher than that of the drooling group with both short-read and long-read NGS, while no significant differences in microbial diversities were found between the three saliva-preservation methods. Our study shows mouthwash and simple collection are not inferior to other sample collection and saliva-preservation methods, respectively. This result is promising since the convenience and cost-effectiveness of mouthwash and simple collection can simplify the saliva sample preparation, which would greatly help clinical operators and lab workers.

Long-read sequencing revealing intragenic deletions in exome-negative spastic paraplegias.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness in the lower extremities. To date, a total of 88 types of SPG are known. To diagnose HSP, multiple technologies, including microarray, direct sequencing, multiplex ligation-dependent probe amplification, and short-read next-generation sequencing, are often chosen based on the frequency of HSP subtypes. Exome sequencing (ES) is commonly used. We used ES to analyze ten cases of HSP from eight families. We identified pathogenic variants in three cases (from three different families); however, we were unable to determine the cause of the other seven cases using ES. We therefore applied long-read sequencing to the seven undetermined HSP cases (from five families). We detected intragenic deletions within the SPAST gene in four families, and a deletion within PSEN1 in the remaining family. The size of the deletion ranged from 4.7 to 12.5 kb and involved 1-7 exons. All deletions were entirely included in one long read. We retrospectively performed an ES-based copy number variation analysis focusing on pathogenic deletions, but were not able to accurately detect these deletions. This study demonstrated the efficiency of long-read sequencing in detecting intragenic pathogenic deletions in ES-negative HSP patients.

Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells.

RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR. It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15. We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15, a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol.

INSurVeyor: improving insertion calling from short read sequencing data

Insertions are one of the major types of structural variations and are defined as the addition of 50 nucleotides or more into a DNA sequence. Several methods exist to detect insertions from next-generation sequencing short read data, but they generally have low sensitivity. Our contribution is two-fold. First, we introduce INSurVeyor, a fast, sensitive and precise method that detects insertions from next-generation sequencing paired-end data. Using publicly available benchmark datasets (both human and non-human), we show that INSurVeyor is not only more sensitive than any individual caller we tested, but also more sensitive than all of them combined. Furthermore, for most types of insertions, INSurVeyor is almost as sensitive as long reads callers. Second, we provide state-of-the-art catalogues of insertions for 1047 Arabidopsis Thaliana genomes from the 1001 Genomes Project and 3202 human genomes from the 1000 Genomes Project, both generated with INSurVeyor. We show that they are more complete and precise than existing resources, and important insertions are missed by existing methods.

Haplotyping Using Long-Range PCR and Nanopore Sequencing to Phase Variants: Lessons Learned From the ABCA4 Locus

Short-read next-generation sequencing has revolutionized our ability to identify variants underlying inherited diseases; however, it does not allow the phasing of variants to clarify their diagnostic interpretation. The advent of widespread, increasingly accurate long-read sequencing has opened up new applications not currently available through short-read next-generation sequencing. One such use is the ability to phase variants to clarify their diagnostic interpretation and to investigate the increasingly prevalent role of cis-acting variants in the pathogenesis of the inherited disease, so-called complex alleles. Complex alleles are becoming an increasingly prevalent part of the study of genes associated with inherited diseases, for example, in ABCA4-related diseases. We sought to establish a cost-effective method to phase contiguous segments of the 130-kb ABCA4 locus by long-read sequencing of overlapping amplification products. Using the comprehensively characterized CEPH sample, NA12878, we verified the accuracy and robustness of our assay. However, in-field assessment of its utility using clinical test cases was hampered by the paucity and distribution of identified variants and by PCR chimerism, particularly where the number of PCR cycles was high. Despite this, we were able to construct robust phase blocks of up to 94.9 kb, representing 73% of the ABCA4 locus. We conclude that, although haplotype analysis of variants located within discrete amplification products was robust and informative, the stitching together of larger phase blocks using overlapping single-molecule reads remained practically challenging.

Diagnostic yield of genetic screening in a diverse, community-ascertained cohort

BackgroundPopulation screening for genetic risk of adult-onset preventable conditions has been proposed as an attractive public health intervention. Screening unselected individuals can identify many individuals who will not be identified through current genetic testing guidelines.MethodsWe sought to evaluate enrollment in and diagnostic yield of population genetic screening in a resource-limited setting among a diverse population. We developed a low-cost, short-read next-generation sequencing panel of 25 genes that had 98.4% sensitivity and 99.98% specificity compared to diagnostic panels. We used email invitations to recruit a diverse cohort of patients in the University of Washington Medical Center system unselected for personal or family history of hereditary disease. Participants were sent a saliva collection kit in the mail with instructions on kit use and return. Results were returned using a secure online portal. Enrollment and diagnostic yield were assessed overall and across race and ethnicity groups.ResultsOverall, 40,857 people were invited and 2889 (7.1%) enrolled. Enrollment varied across race and ethnicity groups, with the lowest enrollment among African American individuals (3.3%) and the highest among Multiracial or Other Race individuals (13.0%). Of 2864 enrollees who received screening results, 106 actionable variants were identified in 103 individuals (3.6%). Of those who screened positive, 30.1% already knew about their results from prior genetic testing. The diagnostic yield was 74 new, actionable genetic findings (2.6%). The addition of more recently identified cancer risk genes increased the diagnostic yield of screening.ConclusionsPopulation screening can identify additional individuals that could benefit from prevention, but challenges in recruitment and sample collection will reduce actual enrollment and yield. These challenges should not be overlooked in intervention planning or in cost and benefit analysis.

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.

Gap-free nuclear and mitochondrial genomes of Fusarium verticillioides strain HN2

Fusarium ear rot (FER) and Fusarium stalk rot (FSR) caused by the filamentous fungus Fusarium verticillioides have become increasingly serious around the world. Additionally, fumonisins produced by F. verticillioides threaten food and feed security. By adding the contribution of genomic resources to better understand the pathosystem including the mechanisms of F. verticillioides–maize interactions, and further improving the quality of the F. verticillioides genome, the gap-free nuclear genome and mitochondrial genome of F. verticillioides strain HN2 were sequenced and assembled. Using Oxford Nanopore long reads and next-generation sequencing short reads, the final 42.81-Mb genome was assembled into 12 contigs (N50 = 4.16-Mb). A total of 13,466 protein-coding genes were annotated, including 1,076 secreted proteins that contain 342 candidate effectors. In addition, we assembled the complete 53,764 bp mitochondrial genome. F. verticillioides strain 7600 genome assemblies are fragmented and high-quality reference genomes were needed. The genomes presented here will serve as an important resource for F. verticillioides research.

An assessment of bioinformatics tools for the detection of human endogenous retroviral insertions in short-read genome sequencing data.

There is a growing interest in the study of human endogenous retroviruses (HERVs) given the substantial body of evidence that implicates them in many human diseases. Although their genomic characterization presents numerous technical challenges, next-generation sequencing (NGS) has shown potential to detect HERV insertions and their polymorphisms in humans. Currently, a number of computational tools to detect them in short-read NGS data exist. In order to design optimal analysis pipelines, an independent evaluation of the available tools is required. We evaluated the performance of a set of such tools using a variety of experimental designs and datasets. These included 50 human short-read whole-genome sequencing samples, matching long and short-read sequencing data, and simulated short-read NGS data. Our results highlight a great performance variability of the tools across the datasets and suggest that different tools might be suitable for different study designs. However, specialized tools designed to detect exclusively human endogenous retroviruses consistently outperformed generalist tools that detect a wider range of transposable elements. We suggest that, if sufficient computing resources are available, using multiple HERV detection tools to obtain a consensus set of insertion loci may be ideal. Furthermore, given that the false positive discovery rate of the tools varied between 8% and 55% across tools and datasets, we recommend the wet lab validation of predicted insertions if DNA samples are available.

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum.

Next-generation sequencing (NGS) is a useful molecular diagnostic tool for genetic diseases. However, due to the presence of highly homologous pseudogenes, it is challenging to use short-read NGS for analyzing mutations of the Shwachman-Bodian-Diamond syndrome (SBDS) gene. The SBDS mutation spectrum was analyzed in the Chinese population, which revealed that SBDS variants were primarily from sequence exchange between SBDS and its pseudogene at the base-pair level, predominantly in the coding region and splice junction of exon two. The c.258+2T>C and c.185_184TA>GT variants were the two most common pathogenic SBDS variants in the Chinese population, resulting in a total carrier frequency of 1.19%. When analyzing pathogenic variants in the SBDS gene from the NGS data, the misalignment was identified as a common issue, and there were different probabilities of misalignment for different pathogenic variants. Here, we present a novel mathematical method for identifying pathogenic variants in the SBDS gene from the NGS data, which utilizes read-depth of the paralogous sequence variant (PSV) loci of SBDS and its pseudogene. Combined with PCR and STR orthogonal experiments, SBDS gene mutation analysis results were improved in 40% of clinical samples, and various types of mutations such as homozygous, compound heterozygous, and uniparental diploid were explored. The findings effectively reduce the impact of misalignment in NGS-based SBDS mutation analysis and are helpful for the clinical diagnosis of SBDS-related diseases, the research into population variation, and the carrier screening.

Molecular Epidemiology of Escherichia coli with Resistance against Third-Generation Cephalosporines Isolated from Deployed German Soldiers-A Retrospective Assessment after Deployments to the African Sahel Region and Other Sites between 2007 and 2016.

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant Escherichia coli isolated between 2007 and 2016 from German soldiers after deployments, with a particular focus on the African Sahel region. A total of 51 third-generation cephalosporine-resistant E. coli isolated from 51 military returnees from deployment collected during the assessment period between 2007 and 2016 were subjected to short-read next-generation sequencing analysis. Returnees from the Sahel region (Djibouti, Mali, South Sudan, Sudan, Sudan, and Uganda) comprised a proportion of 52.9% (27/51). Repeatedly isolated sequence types according to the Warwick University scheme from returnees from the Sahel region were ST38, ST131, and ST648, confirming previous epidemiological assessments from various sub-Saharan African regions. Locally prevalent resistance genes in isolates from returnees from the Sahel region associated with third-generation resistance were blaCTX-M-15, blaCTX-M-27, blaCTX-M-1, blaTEM-169, blaCTX-M-14, blaCTX-M-99-like, blaCTX-M-125, blaSHV-12, and blaDHA-1, while virulence genes were east1, sat, and tsh in declining order of frequency of occurrence each. In line with phenotypically observed high resistance rates for aminoglycosides and trimethoprim/sulfamethoxazole, multiple associated resistance genes were observed. A similar, slightly more diverse situation was recorded for the other deployment sites. In summary, this assessment provides first next-generation sequencing-based epidemiological data on third-generation cephalosporine-resistant E. coli imported by deployed German soldiers with a particular focus on deployments to the Sahel region, thus serving as a small sentinel. The detected sequence types are well in line with the results from previous epidemiological assessments in sub-Saharan Africa.

Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing.

With the development and reduced costs of high-throughput sequencing technology, environmental dark matter, such as novel metagenome-assembled genomes (MAGs) and viruses, is now being discovered easily. However, due to read length limitations, MAGs and viromes often suffer from genome discontinuity and deficiencies in key functional elements. Here, by applying long-read sequencing technology to sediment samples from a Tibetan saline lake, we comprehensively analyzed the performance of high-fidelity (HiFi) reads and the possibility of integration with short-read next-generation sequencing (NGS) data. In total, 207 full-length nonredundant 16S rRNA gene sequences and 19 full-length nonredundant 18S rRNA genes were directly obtained from HiFi reads, which greatly surpassed the retrieval performance of NGS technology. We carried out a cross-sectional comparison among multiple assembly strategies, referred to as 'NGS', 'Hybrid (NGS+HiFi)', and 'HiFi'. Two MAGs and 29 viruses with circular genomes were reconstructed using HiFi reads alone, indicating the great power of the 'HiFi' approach to assemble high-quality microbial genomes. Among the 3 strategies, the 'Hybrid' approach produced the highest number of medium/high-quality MAGs and viral genomes, while the ratio of MAGs containing 16S rRNA genes was significantly improved in the 'HiFi' assembly results. Overall, our study provides a practical metagenomic resolution for analyzing complex environmental samples by taking advantage of both the short-read and HiFi long-read sequencing methods to extract the maximum amount of information, including data on prokaryotes, eukaryotes, and viruses, via the 'Hybrid' approach. IMPORTANCE To expand the understanding of microbial dark matter in the environment, we did the first comparative evaluation of multiple assembly strategies based on high-throughput short-read and HiFi data from lake sediments metagenomic sequencing. The results demonstrated great improvement of the 'Hybrid' assembly method (short-read next-generation sequencing data plus HiFi data) in the recovery of medium/high-quality MAGs and viral genomes. Further analysis showed that HiFi data is important to retrieve the complete circular prokaryotic and viral genomes. Meanwhile, hundreds of full-length 16S/18S rRNA genes were assembled directly from HiFi data, which facilitated the species composition studies of complex environmental samples, especially for understanding micro-eukaryotes. Therefore, the application of the latest HiFi long-read sequencing could greatly improve the metagenomic assembly integrity and promote environmental microbiome research.

A template for multitudes: Germline immune polymorphism of the T cell receptor loci

Short-read next-generation sequencing has failed to adequately genotype the Tcell receptor (TCR) loci, limiting our ability to characterize the role of germline TCR variation. In this issue of Cell Genomics, Rodriguez etal.1 describe how a probe-based hybrid capture approach coupled with long-read sequencing can resolve fully phased TCR locus haplotypes from diploid human genomes.