Reduction of bitter taste receptor gene family in folivorous colobine primates relative to omnivorous cercopithecine primates.

Bitter taste perception is important in preventing animals from ingesting potentially toxic compounds. Whole-genome assembly (WGA) data have revealed that bitter taste receptor genes (TAS2Rs) comprise a multigene family with dozens of intact and disrupted genes in primates. However, publicly available WGA data are often incomplete, especially for multigene families. In this study, we employed a targeted capture (TC) approach specifically probing TAS2Rs for ten species of cercopithecid primates with diverse diets, including eight omnivorous cercopithecine species and two folivorous colobine species. We designed RNA probes for all TAS2Rs that we modeled to be intact in the common ancestor of cercopithecids ("ancestral-cercopithecid TAS2R gene set"). The TC was followed by short-read and high-depth massive-parallel sequencing. TC retrieved more intact TAS2R genes than found in WGA databases. We confirmed a large number of gene "births" at the common ancestor of cercopithecids and found that the colobine common ancestor and the cercopithecine common ancestor had contrasting trajectories: four gene "deaths" and three gene births, respectively. The number of intact TAS2R genes was markedly reduced in colobines (25-28 detected via TC and 20-26 detected via WGA analysis) as compared with cercopithecines (27-36 via TC and 19-30 via WGA). Birth or death events occurred at almost every phylogenetic-tree branch, making the composition of intact genes variable among species. These results show that evolutionary change in intact TAS2R genes is a complex process, refute a simple general prediction that herbivory favors more TAS2R genes, and have implications for understanding dietary adaptations and the evolution of detoxification abilities.

The effect of missing data on evolutionary analysis of sequence capture bycatch, with application to an agricultural pest.

Sequence capture is a genomic technique that selectively enriches target sequences before high throughput next-generation sequencing, to generate specific sequences of interest. Off-target or 'bycatch' data are often discarded from capture experiments, but can be leveraged to address evolutionary questions under some circumstances. Here, we investigated the effects of missing data on a variety of evolutionary analyses using bycatch from an exon capture experiment on the global pest moth, Helicoverpa armigera. We added > 200 new samples from across Australia in the form of mitogenomes obtained as bycatch from targeted sequence capture, and combined these into an additional larger dataset to total > 1000 mitochondrial cytochrome c oxidase subunit I (COI) sequences across the species' global distribution. Using discriminant analysis of principal components and Bayesian coalescent analyses, we showed that mitogenomes assembled from bycatch with up to 75% missing data were able to return evolutionary inferences consistent with higher coverage datasets and the broader literature surrounding H. armigera. For example, low-coverage sequences broadly supported the delineation of two H. armigera subspecies and also provided new insights into the potential for geographic turnover among these subspecies. However, we also identified key effects of dataset coverage and composition on our results. Thus, low-coverage bycatch data can offer valuable information for population genetic and phylodynamic analyses, but caution is required to ensure the reduced information does not introduce confounding factors, such as sampling biases, that drive inference. We encourage more researchers to consider maximizing the potential of the targeted sequence approach by examining evolutionary questions with their off-target bycatch where possible-especially in cases where no previous mitochondrial data exists-but recommend stratifying data at different genome coverage thresholds to separate sampling effects from genuine genomic signals, and to understand their implications for evolutionary research.

Targeted enrichment of whole-genome SNPs from highly burned skeletal remains.

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole-genome single-nucleotide polymorphism (SNP) quality and quantity using next-generation sequencing (NGS). We present whole-genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double-stranded DNA libraries then enriched for whole-genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed-effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole-genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification.

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene ßglu-1 with large variations in white spruce but not Norway spruce.

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and ßglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of ßglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of ßglu-1 and Ugt5 genes. We observed very large copy numbers of ßglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of ßglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

Four conserved amino acids on human papillomavirus E6 predict clinical high-risk types.

Human papillomavirus (HPV) types included in the genus alpha papillomavirus (alpha-HPVs) are subdivided into high- and low-risk HPVs associated with tumorigenicity. According to conventional risk classification, over 30 alpha-HPVs remain unclassified and HPV groups phylogenetically classified using the L1 gene do not exactly correspond to the conventional risk classification groups. Here, we propose a novel cervical lesion progression risk classification strategy. Using four E6 risk distinguishable amino acids (E6-RDAAs), we successfully expanded the conventional classification to encompass alpha-HPVs and resolve discrepancies. We validated our classification system using alpha-HPV-targeted sequence data of 325 cervical swab specimens from participants in Japan. Clinical outcomes significantly correlated with the E6-RDAA classification. Four of five HPV types in the data set that were not conventionally classified (HPV30, 34, 67, and 69) were high-risk according to our classification criteria. This report sheds light on the carcinogenicity of rare genital HPV types using a novel risk classification strategy.

Description of a new species of black coral in the family Aphanipathidae (Anthozoa, Antipatharia) from Puerto Rico.

Black corals (Anthozoa: Antipatharia) are an anthozoan lineage in the class Hexacorallia that occur across a wide range of habitats from the tropics to the poles and from surface waters to depths deeper than 8000 m. A new species of black coral, Aphanipathespuertoricoensissp. nov., collected with a remotely operated vehicle 357 m deep off Puerto Rico is recognized in the family Aphanipathidae. The new species is characterized by very long and loosely coiled primary branches and up to 0.5 mm tall spines with as many as 40 or more small conical tubercles. A phylogeny composed of 13 taxa that are closely related to the new species was reconstructed from 793 nuclear loci to show their systematic relationships. Our study integrated morphological and genomic data to show that this new species is distinct from other species in the genus Aphanipathes. Furthermore, our results add to the growing knowledge of black coral diversity, while further demonstrating the need for exploration in deep waters of the Caribbean Sea.

Telomere integrated scoring system of myelodysplastic syndrome.

INTRODUCTION: Recently, multigene target sequencing is widely performed for the purpose of prognostic prediction and application of targeted therapy. Here, we proposed a new scoring system that encompasses gene variations, telomere length, and Revised International Prognostic Scoring System (IPSS-R) together in Asian myelodysplastic syndrome. METHODS: We developed a new scoring model of these variables: age ≥ 65 years + IPSS-R score + ASXL1 mutation + TP53 mutation + Telomere length (<5.37). According to this new scoring system, patients were divided into four groups: very good score cutoff (≤3.0), good (3.0-4.5), poor (4.5-7.0), and very poor (>7.0). RESULTS: The median OS was 170.1, 100.4, 46.0, and 12.0 months for very good, good, poor, and very poor, retrospectively (p < 0.001). Meanwhile, according to the conventional IPSS-R scoring system, the median OS was 141.3, 50.2, 93.0, 36.0, and 16.2 months for very low, low, intermediate, high, and very high, retrospectively (p < 0.001). CONCLUSIONS: The newly developed model incorporating molecular variations and TL yielded more clear separations of the survival curves. By adding the presence of gene mutation and telomere length to the existing IPSS-R, its predictive ability can be further improved in myelodysplastic syndrome.

Linking breadfruit cultivar names across the globe connects histories after 230 years of separation.

Every crop has a story. The story of breadfruit (Artocarpus altilis), an increasingly valued staple crop in tropical agroforestry systems, is filled with intrigue, oppression, and remains incomplete. The Caribbean is a major producer and consumer of breadfruit, yet most breadfruit there came from a single 1793 introduction aimed at providing a cheap food source for slaves forced to work on British plantations. St. Vincent was the first significant point of Caribbean introduction and played a vital role in subsequent breadfruit distribution throughout the region. Hundreds of cultivars are documented in breadfruit's native Oceania. It remains a mystery, however, which ones were introduced to the Caribbean 230 years ago-still comprising the vast diversity found there today. Integrating local knowledge, historical documents and specimens, morphological data, and DNA, we identify eight major global breadfruit lineages-five of which are found in the Caribbean and likely represent the original 1793 introduction. Genetic data were able to match two Caribbean cultivar names confidently to their Oceania counterparts. Genetics and morphology together enabled additional possible matches. Many other named cultivars within lineages are too genetically similar to differentiate, highlighting difficulties of defining and identifying variation among clonally propagated triploid crops. Breadfruit is important in resilient agroforestry in tropical islands predicted to be especially affected by climate change. Findings reveal global links, building upon collective knowledge that can be used to inform breadfruit management. Results are also summarized in a brochure about breadfruit history and diversity in St. Vincent, and the Caribbean more broadly.

Application of targeted capture high depth sequencing and transcriptome sequencing in childhood B-cell acute lymphoblastic leukemia / 白血病·淋巴瘤

Objective:To investigate the differences among targeted capture high depth sequencing (Panel-seq), transcriptome sequencing (RNA-seq) and traditional detection methods in cytogenetic and molecular genetic typing of childhood B-cell acute lymphoblastic leukemia (B-ALL) and their significances.Methods:The clinical data of 152 newly diagnosed childhood B-ALL cases in Guangzhou Women and Children's Medical Center from September 2020 to December 2021 were retrospectively analyzed. Along with traditional cytogenetic and molecular detection methods including karyotyping, fluorescence in situ hybridization (FISH) and 43 kinds of fusion gene quantitative screening for traditional cells and molecular genetic detection, both Panel-seq and RNA-seq were also performed. Panel-seq covered more than 600 genes with common mutations in hematological tumors, from which fusion genes and gene mutations were both analyzed. RNA-seq was used to analyze fusion genes, gene mutations, gene expression, and copy number variation at the chromosome level. High hyperdiploid karyotype was estimated by using gene expression profile clustering and copy number variations. The cytogenetic typing results of all detection methods were also analyzed.Results:Among 152 patients, 93 cases were males and 59 cases were females, with the median age of 4.0 years (0.8-13.0 years). The median blast cell ratio was 0.855 (0.215-0.965). The traditional detection methods could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusion, 27 cases (17.8%) with ETV6-RUNX1, 1 case (0.7%) with iAMP21, 5 cases (3.3%) with MLL rearrangement, 8 cases (5.3%) with TCF3-PBX1 and 22 cases (14.5%) with high hyperdiploid karyotype. Panel-seq could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusions, 27 cases (17.8%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 5 cases (3.3%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 8 cases (5.3%) with TCF3-PBX1 fusions, 4 cases (2.6%) with ZNF384 gene-related fusions, and 2 cases (1.3%) with IKZF1 N159Y mutations. Among 152 patients, 1 case with MLL rearrangement didn't receive RNA-seq detection because of sample quality; in other 151 B-ALL cases, 1 case (0.7%) with ACIN1-NUTM1, 4 cases (2.6%) with BCR-ABL1, 3 cases (2.0%) with CRLF2 gene-related fusions, 8 cases (5.3%) with DUX4 gene-related fusions, 27 cases (17.9%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 4 cases (2.6%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 1 case (0.7%) with ZMIZ1-ABL1, 8 cases (5.3%) with TCF3-PBX1,4 cases (2.6%) with ZNF384 gene-related fusions, 61 cases (40.4%) with hyperdiploid karyotypes, and 2 cases (1.3%) with IKZF1 N159Y mutations were detected; RNA-seq had obvious advantage in detecting fusion gene and hyperdiploid karyotype. The cytogenetic and molecular genetic typing rates of traditional method, Panel-seq and RNA-seq were 45.4% (69/152), 40.1% (61/152) and 87.4% (132/151), respectively. The combination of the three could identify 89.5% (136/152) of childhood B-ALL patients.Conclusions:The combination of Panel-seq and RNA-seq can increase the detection rate of genetic abnormality in childhood B-ALL, which provides a more accurate molecular genetic classification for B-ALL and the basis for treatment guideline and prognosis judgement.

Targeted screening of genetic associations with COVID-19 susceptibility and severity.

The COVID-19 pandemic has resulted in great morbidity and mortality worldwide and human genetic factors have been implicated in the susceptibility and severity of COVID-19. However, few replicate researches have been performed, and studies on associated genes mainly focused on genic regions while regulatory regions were a lack of in-depth dissection. Here, based on previously reported associated variants and genes, we designed a capture panel covering 1,238 candidate variants and 25 regulatory regions of 19 candidate genes and targeted-sequenced 96 mild and 145 severe COVID-19 patients. Genetic association analysis was conducted between mild and severe COVID-19 patients, between all COVID-19 patients and general population, or between severe COVID-19 patients and general population. A total of 49 variants were confirmed to be associated with susceptibility or severity of COVID-19 (p < 0.05), corresponding to 18 independent loci. Specifically, rs1799964 in the promoter of inflammation-related gene TNF, rs9975538 in the intron of interferon receptor gene IFNAR2, rs429358 in the exon of APOE, rs1886814 in the intron of FOXP4-AS1 and a list of variants in the widely reported 3p21.31 and ABO gene were confirmed. It is worth noting that, for the confirmed variants, the phenotypes of the cases and controls were highly consistent between our study and previous reports, and the confirmed variants identified between mild and severe patients were quite different from those identified between patients and general population, suggesting the genetic basis of susceptibility and severity of SARS-CoV-2 infection might be quite different. Moreover, we newly identified 67 significant associated variants in the 12 regulatory regions of 11 candidate genes (p < 0.05). Further annotation by RegulomeDB database and GTEx eQTL data filtered out two variants (rs11246060 and rs28655829) in the enhancer of broad-spectrum antiviral gene IFITM3 that might affect disease severity by regulating the gene expression. Collectively, we confirmed a list of previously reported variants and identified novel regulatory variants associated with susceptibility and severity of COVID-19, which might provide biological and clinical insights into COVID-19 pathogenesis and treatment.

Capturing the antibiotic resistome of preterm infants reveals new benefits of probiotic supplementation.

BACKGROUND: Probiotic use in preterm infants can mitigate the impact of antibiotic exposure and reduce rates of certain illnesses; however, the benefit on the gut resistome, the collection of antibiotic resistance genes, requires further investigation. We hypothesized that probiotic supplementation of early preterm infants (born < 32-week gestation) while in hospital reduces the prevalence of antibiotic resistance genes associated with pathogenic bacteria in the gut. We used a targeted capture approach to compare the resistome from stool samples collected at the term corrected age of 40 weeks for two groups of preterm infants (those that routinely received a multi-strain probiotic during hospitalization and those that did not) with samples from full-term infants at 10 days of age to identify if preterm birth or probiotic supplementation impacted the resistome. We also compared the two groups of preterm infants up to 5 months of age to identify persistent antibiotic resistance genes. RESULTS: At the term corrected age, or 10 days of age for the full-term infants, we found over 80 antibiotic resistance genes in the preterm infants that did not receive probiotics that were not identified in either the full-term or probiotic-supplemented preterm infants. More genes associated with antibiotic inactivation mechanisms were identified in preterm infants unexposed to probiotics at this collection time-point compared to the other infants. We further linked these genes to mobile genetic elements and Enterobacteriaceae, which were also abundant in their gut microbiomes. Various genes associated with aminoglycoside and beta-lactam resistance, commonly found in pathogenic bacteria, were retained for up to 5 months in the preterm infants that did not receive probiotics. CONCLUSIONS: This pilot survey of preterm infants shows that probiotics administered after preterm birth during hospitalization reduced the diversity and prevented persistence of antibiotic resistance genes in the gut microbiome. The benefits of probiotic use on the microbiome and the resistome should be further explored in larger groups of infants. Due to its high sensitivity and lower sequencing cost, our targeted capture approach can facilitate these surveys to further address the implications of resistance genes persisting into infancy without the need for large-scale metagenomic sequencing. Video Abstract.

Evolutionary origin of pathogenic GJB2 alleles in China.

The frequency of the pathogenic allele of the autosomal recessive deafness gene GJB2 varies among different populations in the world, and accumulates to a sufficiently high frequency in certain population. The purpose of this study is to investigate the origin and evolution of GJB2 pathogenic alleles in Chinese deaf patients. Children with non-syndromic hearing loss, and their parents, from 295 families were recruited. Customized capture probes targeted at 943 single nucleotide polymorphisms (SNPs) related to GJB2 gene were designed for sequencing of genomic DNA in blood samples. Haplotypes carrying pathogenic allele were analyzed through linkage disequilibrium block building, ancestry tracing, and extended haplotype heterozygosity calculation. Two pathogenic GJB2 alleles, c.235delC (18.41%) and c.109G > A (15.57%), were observed in 867 donors. For c.235delC allele, three different core haplotypes with one major haplotype (97.32%) were found, and their core SNPs were 100% conserved. For c.109G > A allele, six different haplotypes with one major haplotype (93.28%) were found and the major c.109G > A allele evolved from a specific ancestral haplotype. Geographical origins of donors carrying GJB2 c.109G > A and c.235delC core haplotypes centered between Qinghai and Neimenggu. GJB2 c.235delC has long-range linkage disequilibrium. No positive selection signature was found for GJB2 c.235delC or c.109G > A in the studied population. In conclusion, we discovered a single origin of GJB2 c.235delC allele and multiple independent origins of GJB2 c.109G > A allele. Alternative to positive selection or multiple independent recurrent mutation event, population bottleneck effect might account for the observed high population frequency of these pathogenic alleles.

The landscape of genetic aberrations in myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a rare subtype of sarcoma, whose genetic basis is poorly understood. We analyzed 69 MFS cases using whole-genome (WGS), whole-exome (WES) and/or targeted-sequencing (TS). Newly sequenced genomic data were combined with additional deposited 116 MFS samples. WGS identified a high number of structural variations (SVs) per tumor most frequently affecting the TP53 and RB1 loci, 40% of tumors showed a BRCAness-associated mutation signature, and evidence of chromothripsis was found in all cases. Most frequently mutated/copy number altered genes affected known disease drivers such as TP53 (56.2%), CDKN2A/B (29.7%), RB1 (27.0%), ATRX (19.5%) and HDLBP (18.9%). Several previously unappreciated genetic aberrations including MUC17, FLG and ZNF780A were identified in more than 20% of patients. Longitudinal analysis of paired diagnosis and relapse time points revealed a 1.2-fold mutation number increase accompanied with substantial changes in clonal composition over time. Our study highlights the genetic complexity underlying sarcomagenesis of MFS.

Next-Generation DNA Sequencing-Based Gene Panel for Diagnosis and Genetic Risk Stratification in Onco-Hematology.

A suitable diagnostic classification of myeloid neoplasms and acute leukemias requires testing for a large number of molecular biomarkers. Next-generation sequencing is a technology able to integrate identification of the vast majority of them in a single test. This manuscript includes the design, analytical validation and clinical feasibility evaluation of a molecular diagnostic kit for onco-hematological diseases. It is based on sequencing of the coding regions of 76 genes (seeking single-nucleotide variants, small insertions or deletions and CNVs), as well as the search for fusions in 27 target genes. The kit has also been designed to detect large CNVs throughout the genome by including specific probes and employing a custom bioinformatics approach. The analytical and clinical feasibility validation of the Haematology OncoKitDx panel has been carried out from the sequencing of 170 patient samples from 6 hospitals (in addition to the use of commercial reference samples). The analytical validation showed sensitivity and specificity close to 100% for all the parameters evaluated, with a detection limit of 2% for SNVs and SVs, and 20% for CNVs. Clinically relevant mutations were detected in 94% of all patients. An analysis of the correlation between the genetic risk classification of AML (according to ELN 2017) established by the hospitals and that obtained by the Haematology OncoKitDx panel showed an almost perfect correlation (K = 0.94). Among the AML samples with a molecular diagnosis, established by the centers according to the WHO, the Haematology OncoKitDx analysis showed the same result in 97% of them. The panel was able to adequately differentiate between MPN subtypes and also detected alterations that modified the diagnosis (FIP1L1-PDGFRA). Likewise, the cytogenetic risk derived from the CNV plot generated by the NGS panel correlated substantially with the results of the conventional karyotype (K = 0.71) among MDS samples. In addition, the panel detected the main biomarkers of prognostic value among patients with ALL. This validated solution enables a reliable analysis of a large number of molecular biomarkers from a DNA sample in a single assay.

Combined genetic screening and traditional biochemical screening to optimize newborn screening systems.

Newborn screening can detect around 40 different diseases based on biochemical indicators and has resulted in the improved quality of life for children suffering from genetic diseases. However, NBS is limited as it does not cover all genetic diseases in newborns and has high rates of false positives and negatives. Genetic screening can be used to address the shortcomings of traditional biochemical screening, however, the comprehensive clinical value of genetic screening is yet to be systematically studied. In this study, we used two different genetic screening methods to examine 200 cases of NBS. We found that genetic screening can be used to identify a broader spectrum of diseases and is not limited to traditional biochemical screening diseases; it can identify positive cases of disease and can eliminate false positives caused by multiple factors such as pathogenic variants carrier or the mode of childbirth. Genetic screening has shortened the time to diagnosis and reduced the costs of testing. Furthermore, we found that the biochemical detection results were limited when patients simultaneously carried multiple pathogenic mutations. Our research provisionally demonstrates the necessity, feasibility and significance of clinical genetic screening in newborns and provides a solid basis for future clinical developments.

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

A dense linkage map for a large repetitive genome: discovery of the sex-determining region in hybridizing fire-bellied toads (Bombina bombina and Bombina variegata).

Genomic analysis of hybrid zones offers unique insights into emerging reproductive isolation and the dynamics of introgression. Because hybrid genomes consist of blocks inherited from one or the other parental taxon, linkage information is essential. In most cases, the spectrum of local ancestry tracts can be efficiently uncovered from dense linkage maps. Here, we report the development of such a map for the hybridizing toads, Bombina bombina and Bombina variegata (Anura: Bombinatoridae). Faced with the challenge of a large (7-10 Gb), repetitive genome, we set out to identify a large number of Mendelian markers in the nonrepetitive portion of the genome that report B. bombina vs B. variegata ancestry with appropriately quantified statistical support. Bait sequences for targeted enrichment were selected from a draft genome assembly, after filtering highly repetitive sequences. We developed a novel approach to infer the most likely diplotype per sample and locus from the raw read mapping data, which is robust to over-merging and obviates arbitrary filtering thresholds. Validation of the resulting map with 4755 markers underscored the large-scale synteny between Bombina and Xenopus tropicalis. By assessing the sex of late-stage F2 tadpoles from histological sections, we identified the sex-determining region in the Bombina genome to 7 cM on LG5, which is homologous to X. tropicalis chromosome 5, and inferred male heterogamety. Interestingly, chromosome 5 has been repeatedly recruited as a sex chromosome in anurans with XY sex determination.

Morphological and molecular description of a new genus and species of black coral (Cnidaria: Anthozoa: Hexacorallia: Antipatharia: Antipathidae: Blastopathes) from Papua New Guinea.

Blastopathes medusa gen. nov., sp. nov., is described from Kimbe Bay, Papua New Guinea, based on morphological and molecular data. Blastopathes, assigned to the Antipathidae, is a large, mythology-inspiring black coral characterized by clusters of elongate stem-like branches that extend out at their base and then curve upward. Colonies are not pinnulate and contain single branches, which could represent new branch cluster formations. Morphological and molecular (mitochondrial DNA and targeted capture of nuclear loci) evidence supporting the establishment of a new genus is discussed. This is the first study to utilize the target capture of ultraconserved elements (UCEs) and exonic loci to elucidate phylogenetic relationships among black corals and to identify and place a new genus and species.

Comprehensive Determination of Mycobacterium tuberculosis and Nontuberculous Mycobacteria From Targeted Capture Sequencing.

Infection of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) challenges effective pulmonary infectious disease control. Current phenotypic and molecular assays could not comprehensively and accurately diagnose MTB, NTM, and drug resistance. Next-generation sequencing allows an "all-in-one" approach providing results on expected drug susceptibility testing (DST) and the genotype of NTM strains. In this study, targeted capture sequencing was used to analyze the genetic backgrounds of 4 MTB strains and 32 NTM pathogenic strains in 30 clinical samples, including 14 sputum specimens and 16 bronchoalveolar lavage fluid samples. Through comparing with other TB diagnostic tests, we proved that targeted capture sequencing could be used as a highly sensitive (91.3%) and accurate (83.3%) method to diagnose TB, as well as MGIT 960. Also, we identified 7 NTM strains in 11 patients; among them, seven patients were MTB/NTM co-affected, which indicated that it was a meaningful tool for the diagnosis and treatment of NTM infection diseases in clinic. However, based on a drug-resistant mutation library (1,325 drug resistance loci), only 9 drug resistance strains and 22 drug resistance loci were discovered, having considerable discordance with the drug-resistant results of MGIT 960. Our finding indicated that targeted capture sequencing approach was applicable for the comprehensive and accurate diagnosis of MTB and NTM. However, from data presented here, the DST results identified by next-generation sequencing (NGS) showed a relatively low consistency with MGIT 960, especially in sputum samples. Further work should be done to explore the reasons for low drug-resistance detection rate of NGS.

Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?

BACKGROUND: Next generation sequencing (NGS) can recover DNA data from valuable extant and extinct museum specimens. However, archived or preserved DNA is difficult to sequence because of its fragmented, damaged nature, such that the most successful NGS methods for preserved specimens remain sub-optimal. Improving wet-lab protocols and comprehensively determining the effects of sample age on NGS library quality are therefore of vital importance. Here, I examine the relationship between sample age and several indicators of library quality following targeted NGS sequencing of ~ 1300 loci using 271 samples of pinned moth specimens (Helicoverpa armigera) ranging in age from 5 to 117 years. RESULTS: I find that older samples have lower DNA concentrations following extraction and thus require a higher number of indexing PCR cycles during library preparation. When sequenced reads are aligned to a reference genome or to only the targeted region, older samples have a lower number of sequenced and mapped reads, lower mean coverage, and lower estimated library sizes, while the percentage of adapters in sequenced reads increases significantly as samples become older. Older samples also show the poorest capture success, with lower enrichment and a higher improved coverage anticipated from further sequencing. CONCLUSIONS: Sample age has significant, measurable impacts on the quality of NGS data following targeted enrichment. However, incorporating a uracil-removing enzyme into the blunt end-repair step during library preparation could help to repair DNA damage, and using a method that prevents adapter-dimer formation may result in improved data yields.