Explore the vast range of titles available.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

The island of Sulawesi is the largest in Indonesian Wallacea, one of the most important ecoregions in SE Asia and globally. Here, we generate a comprehensive and detailed map of forest type, its condition, and some of its threats, which highlights key forest conservation areas, pinpoints frontlines within them, and provides the basis for the development of more specific objectives. We relied upon a variety of techniques to generate five main descriptors of forest quality: condition, its level of endangerment, its landscape setting, its simulated fate given a simple model of forest change, and its overall size. Using the results of this analysis, we assessed the existing protected areas (PA), recognized by the Indonesian government, and a conservation portfolio (CP) generated by a recently completed Ecoregional Conservation Assessment (ECA). Our map of conservation priorities is congruent with previous conservation activities, although several priority areas were identified outside of the current PA system and should be the focus of strategic protected area development. Our ranking system is simple, transparent, and flexible. Its modular construction will allow local managers to choose among available proxy measures and to add their own conservation values according to specific priorities and desired outcomes. We envision this analysis as the foundation upon which more specific conservation strategies, based upon detailed biotic information as it becomes available, can be developed.

Direct analysis of unassembled genomic data could greatly increase the power of short read DNA sequencing technologies and allow comparative genomics of organisms without a completed reference available. Here, we compare 174 chloroplasts by analyzing the taxanomic distribution of short kmers across genomes [1]. We then assemble de novo contigs centered on informative variation. The localized de novo contigs can be separated into two major classes: tip = unique to a single genome and group = shared by a subset of genomes. Prior to assembly, we found that ~18% of the chloroplast was duplicated in the inverted repeat (IR) region across a four-fold difference in genome sizes, from a highly reduced parasitic orchid [2] to a massive algal chloroplast [3], including gnetophytes [4] and cycads [5]. The conservation of this ratio between single copy and duplicated sequence was basal among green plants, independent of photosynthesis and mechanism of genome size change, and different in gymnosperms and lower plants. Major lineages in the angiosperm clade differed in the pattern of shared kmers and de novo contigs. For example, parasitic plants demonstrated an expected accelerated overall rate of evolution, while the hemi-parasitic genomes contained a great deal more novel sequence than holo-parasitic plants, suggesting different mechanisms at different stages of genomic contraction. Additionally, the legumes are diverging more quickly and in different ways than other major families. Small duplicated fragments of the rrn23 genes were deeply conserved among seed plants, including among several species without the IR regions, indicating a crucial functional role of this duplication. Localized de novo assembly of informative kmers greatly reduces the complexity of large comparative analyses by confining the analysis to a small partition of data and genomes relevant to the specific question, allowing direct analysis of next-gen sequence data from previously unstudied genomes and rapid discovery of informative candidate regions.

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

The visual-recipient sector of the cat striatum receives corticostriate input from over 15 higher visual and oculomotor-related areas of the cortex and appears homologous with the physiologically characterized region of mixed visual and oculomotor inputs within the primate caudate nucleus. This area in the cat involves the dorsolateral caudate and a strip of the caudal putamen. In a first series of experiments, the former was injected with a retrograde tracer in several cats. Thalamostriate cells were found in extensive regions, including the intralaminar nuclei, certain motor-related nuclei, and, most notably, across much of the extrageniculate visual thalamus. In another set of experiments, anterograde tracers were also injected into the superior colliculus (SC), and labeled tectothalamic fibers were observed in all thalamic sites projecting to the visual-recipient striatum. These findings highlight for the first time the need for the SC to be considered in models of thalamostriate and visual/oculomotor-striatal function(s). Moreover, the data bring to light the fact that basal-ganglia outflow reaching the SC via striatonigro-nigrotectal circuitry is well positioned to modulate ascending tecto-thalamic-thalamostriatal signals destined for the visual-recipient striatum.

Pharmacogenomics is central to precision medicine, informing medication safety and efficacy. Pharmacogenomic diplotyping of complex genes requires full-length DNA sequences and detection of structural rearrangements. We introduce StarPhase, a tool that leverages PacBio HiFi sequence data to diplotype 21 CPIC Level A pharmacogenes and provides detailed haplotypes and supporting visualizations for , , and . StarPhase diplotypes have high concordance with benchmarks where 99.5% are either exact matches or minor discrepancies. Manual inspection of the 0.5% mismatches indicates they were correctly called by StarPhase. With StarPhase, we update or correct 26.2% of GeT-RM pharmacogenomic diplotypes. Population distributions from StarPhase mostly reflect those of the All of Us cohort, while also highlighting gaps in existing pharmacogenomic databases that long-read sequencing can fill. With a single HiFi whole genome sequencing assay, StarPhase enables robust PGx diplotyping even as additional pharmacogenes and haplotypes are discovered.

High-throughput sequencing provides sufficient means for determining genotypes of clinically important pharmacogenes that can be used to tailor medical decisions to individual patients. However, pharmacogene genotyping, also known as star-allele calling, is a challenging problem that requires accurate copy number calling, structural variation discovery, variant calling and phasing within each pharmacogene copy present in the sample. Here we introduce Aldy 4, a fast and efficient tool for genotyping pharmacogenes that utilizes combinatorial optimization for accurate star-allele calling across different sequencing technologies. Aldy 4 adds support for long reads and ships with a novel phasing model and improved copy number and variant calling models. We compare Aldy 4 against the current state-of-the-art star-allele callers on a large and diverse set of samples and genes sequenced by various sequencing technologies, such as whole-genome and targeted Illumina sequencing, barcoded 10X Genomics and PacBio HiFi. We show that Aldy 4 is the most accurate star-allele caller with near-perfect accuracy in all evaluated contexts. We hope that Aldy remains an invaluable tool in the clinical toolbox even with the advent of long-read sequencing technologies. Aldy 4 is available at https://github.com/0xTCG/aldy.

Spinal muscular atrophy, a leading cause of early infant death, is caused by biallelic mutations of the SMN1 gene. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is impossible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across five ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations and testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7-3.0%). Extending beyond simple copy number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers. Competing Interest Statement X.C., J.H. and M.A.E. are employees of Pacific Biosciences. Footnotes * Supplemental files updated.

Adam Garfinkle's Viewpoints article \"Croats Provide U.S. an Opportunity\" [Aug. 10] is an enlightened, insightful and constructive commentary on the Balkan wars: Enlightened because it eschews sensationalistic demonizing of one side over the others, instead recognizing that any difference in viciousness among Serbs, Muslims and Croats in any Balkan war is strictly a consequence of...