Mb Of Sequence Research Articles

The first high-quality genome assembly and annotation of Anthocidaris crassispina

Anthocidaris crassispina is a very popular edible sea urchin distributed along the coast of the South China Sea. In this study, we performed whole-genome sequencing and generated a chromosome-level assembly of this species. The total length of the genomic contig sequence was 891.02 Mb, and contig N50 was 808.15 kb when Hifiasm was used for assembly. The Hi-C library was constructed and sequenced, yielding approximately 68.61 Gb of data. After Hi-C assembly, approximately 886.72 Mb of sequence was able to be mapped onto 21 chromosomes, accounting for 99.52% of the total genome length. Among the sequences located on the chromosomes, those for which the order and direction could be determined accounted for approximately 826.82 Mb, or 93.24% of the total length. These results provide valuable resources for further study of A. crassispina at the genetic level.

Chromosome-level genome assembly of the cereal cyst nematode Heterodera flipjevi

As an economically important plant parasitic nematode (PPN), Heterodera filipjevi causes great damage on wheat, and now it was widely recorded in many countries. While multiple genomes of PPNs have been published, high-quality genome assembly and annotation on H. filipjevi have yet to be performed. This study presents a chromosome-scale genome assembly and annotation for H. filipjevi, utilizing a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing technologies. The genome consists of 9 pseudo-chromosomes that contain 134.19 Mb of sequence, with a scaffold N50 length of 11.88 Mb. In total, 10,036 genes were annotated, representing 75.20% of the total predicted protein-coding genes. Our study provides the first chromosome-scale genome for H. filipjevi, which is also the inaugural high-quality genome of cereal cyst nematodes (CCNs). It provides a valuable genomic resource for further biological research and pest management of cereal cyst nematodes disease.

Chromosome-scale genomics, metabolomics, and transcriptomics provide insight into the synthesis and regulation of phenols in Vitis adenoclada grapes.

Vitis adenoclada is a wild grape unique to China. It exhibits well resistance to heat, humidity, fungal disease, drought, and soil infertility. Here, we report the high-quality, chromosome-level genome assembly of GH6 (V. adenoclada). The 498.27 Mb genome contained 221.78 Mb of transposable elements, 28,660 protein-coding genes, and 481.44 Mb of sequences associated with 19 chromosomes. GH6 shares a common ancestor with PN40024 (Vitis vinifera) from approximately 4.26-9.01 million years ago, whose divergence occurred later than Vitis rotundifolia and Vitis riparia. Widely-targeted metabolome and transcriptome analysis revealed that the profiles and metabolism of phenolic compounds in V. adenoclada varieties significantly were differed from other grape varieties. Specifically, V. adenoclada varieties were rich in phenolic acids and flavonols, whereas the flavan-3-ol and anthocyanin content was lower compared with other varieties that have V. vinifera consanguinity in this study. In addition, ferulic acid and stilbenes content were associated with higher expressions of COMT and STSs in V. adenoclada varieties. Furthermore, MYB2, MYB73-1, and MYB73-2 were presumably responsible for the high expression level of COMT in V. adenoclada berries. MYB12 (MYBF1) was positively correlated with PAL, CHS, FLS and UFGT.Meanwhile, MYB4 and MYBC2-L1 may inhibit the synthesis of flavan-3-ols and anthocyanins in two V. adenoclada varieties (YN2 and GH6). The publication of the V. adenoclada grape genome provides a molecular foundation for further revealing its flavor and quality characteristics, is also important for identifying favorable genes of the East Asian species for future breeding.

The genome of Aechmea fasciata provides insights into the evolution of tank epiphytic habits and ethylene-induced flowering

Aechmea fasciata is one of the most popular bromeliads and bears a water-impounding tank with a vase-like rosette. The tank habit is a key innovation that has promoted diversity among bromeliads. To reveal the genomic basis of tank habit formation and ethylene-induced flowering, we sequenced the genome of A. fasciata and assembled 352 Mb of sequences into 24 chromosomes. Comparative genomic analysis showed that the chromosomes experienced at least two fissions and two fusions from the ancestral genome of A. fasciata and Ananas comosus. The gibberellin receptor gene GID1C-like was duplicated by a segmental duplication event. This duplication may affect GA signalling and promote rosette expansion, which may permit water-impounding tank formation. During ethylene-induced flowering, AfFTL2 expression is induced and targets the EIN3 binding site ‘ATGTAC’ by AfEIL1-like. The data provided here will serve as an important resource for studying the evolution and mechanisms underlying flowering time regulation in bromeliads.

An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster

BackgroundGenome complexity is largely linked to diversification and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non-duplicated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identification and characterization of complex variability between genomes for plant breeding.ResultsHere we improve and demonstrate the use of CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4.5% and a depth per sample of 11.9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region.ConclusionsCRISPR-Cas9 enrichment is a versatile and powerful tool for long-read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed.

Chromosome-level assembly of Gymnocypris eckloni genome

Gymnocypris eckloni is widely distributed in isolated lakes and the upper reaches of the Yellow River and play significant roles in the trophic web of freshwater communities. In this study, we generated a chromosome-level genome of G. eckloni using PacBio, Illumina and Hi-C sequencing data. The genome consists of 23 pseudo-chromosomes that contain 918.68 Mb of sequence, with a scaffold N50 length of 43.54 Mb. In total, 23,157 genes were annotated, representing 94.80% of the total predicted protein-coding genes. The phylogenetic analysis showed that G. eckloni was most closely related to C. carpio with an estimated divergence time of ~34.8 million years ago. For G. eckloni, we identified a high-quality genome at the chromosome level. This genome will serve as a valuable genomic resource for future research on the evolution and ecology of the schizothoracine fish in the Qinghai-Tibetan Plateau.

Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history.

A cattle pangenome representation was created based on the genome sequences of 898 cattle representing 57 breeds. The pangenome identified 83 Mb of sequence not found in the cattle reference genome, representing 3.1% novel sequence compared with the 2.71-Gb reference. A catalog of structural variants developed from this cattle population identified 3.3 million deletions, 0.12 million inversions, and 0.18 million duplications. Estimates of breed ancestry and hybridization between cattle breeds using insertion/deletions as markers were similar to those produced by single nucleotide polymorphism-based analysis. Hundreds of deletions were observed to have stratification based on subspecies and breed. For example, an insertion of a Bov-tA1 repeat element was identified in the first intron of the APPL2 gene and correlated with cattle breed geographic distribution. This insertion falls within a segment overlapping predicted enhancer and promoter regions of the gene, and could affect important traits such as immune response, olfactory functions, cell proliferation, and glucose metabolism in muscle. The results indicate that pangenomes are a valuable resource for studying diversity and evolutionary history, and help to delineate how domestication, trait-based breeding, and adaptive introgression have shaped the cattle genome.

A de novo genome assembly of Solanum verrucosum Schlechtendal, a Mexican diploid species geographically isolated from other diploid A-genome species of potato relatives.

There are over 100 known species of cultivated potatoes and their wild relatives. Many of these species, including cultivated potatoes, share the A genome; these species are mainly distributed in South America and are reproductively isolated from Mexican diploid species. The only diploid A-genome species distributed in Mexico is Solanum verrucosum Schlechtendal, which is also a maternal progenitor of Mexican polyploid species. In this study, we constructed a high-quality de novo assembly of the S. verrucosum genome using PacBio long-read sequencing and Hi-C scaffolding technologies. A monohaploid clone (2n = x = 12) of S. verrucosum was used to reduce assembly difficulty due to the heterozygous nature of the species. The final sequence assembly consisted of 780.2 Mb of sequence, 684.0 Mb of which were anchored to the 12 chromosomes, with a scaffold N50 of 55.2 Mb. Putative centromeres were identified using publicly available data obtained via chromatin immunoprecipitation sequencing against a centromere-specific histone 3 protein. Transposable elements accounted for approximately 61.8% (482.1 Mb) of the genome, and 46,904 genes were functionally annotated. High gene synteny and similarity were revealed among the genomes of S. verrucosum, Solanum commersonii, Solanum chacoense, Solanum phureja, Solanum tuberosum, and Solanum lycopersicum. The reference-quality S. verrucosum genome will provide new insights into the evolution of Mexican polyploid species and contribute to potato breeding programs.

Investigation of genetic diversity and selection signatures in Czech cattle genetic resources revealed by genome-wide analysis

Preserving genetic diversity and preventing its loss in livestock populations is essential for further sustainable development and breeding. The main purposes of this study were to estimate the current status of genetic diversity of the approved genetic resources (GRs) in indigenous Czech Red (CR) and Czech Spotted (CS) cattle and assess the impact of diversifying selection on two CR subpopulations based on genome-wide single-nucleotide polymorphism (SNP) analysis. The main within-breed genetic diversity indicators of CR and CS (share of polymorphic SNPs, observed heterozygosity, and minor allele frequency) are intermediate or high, and the inbreeding coefficients are below zero. The small degree of genetic differentiation (maximum FST = 0.088) suggests a low divergence between the breeds and populations. Principal component analysis (PCA) shows a less genetic variation in CS and, in contrast, larger variability within and genetic similarity between the two analysed CR subpopulations. Putative selection signatures based on FST revealed that the ten most differentiated genomic regions between the two CR subpopulations were located on chromosomes 1, 3, 11, 12, 18, and 22, encompassing only 7 Mb of sequence and 69 protein-coding genes. Selection differences are low and probably attributed to the influence of the CS genome exploited in CR cattle revitalization. The majority of the candidate genes are involved in the immune system response, fertility, and neuronal and brain function. Gene Ontology enrichment and quantitative trait loci database analyses show that the genes act in milk and meat production and reproductive performance. These findings expand our knowledge about the genomic diversity of endangered Czech indigenous cattle and provide helpful information to organize their breeding programmes and preservation.

Chromosome-level genome sequence assembly and genome-wide association study of Muscadinia rotundifolia reveal the genetics of 12 berry-related traits.

Vitis has two subgenera: Euvitis, which includes commercially important Vitis vinifera and interspecific hybrid cultivars, and Muscadinia. Of note, the market for Muscadinia grapes remains small, and only Muscadinia rotundifolia is cultivated as a commercial crop. To establish a basis for the study of Muscadinia species, we generated chromosome-level whole-genome sequences of Muscadinia rotundifolia cv. Noble. A total of 393.8Mb of sequences were assembled from 20 haploid chromosomes, and 26 394 coding genes were identified from the sequences. Comparative analysis with the genome sequence of V. vinifera revealed a smaller size of the M. rotundifolia genome but highly conserved gene synteny. A genome-wide association study of 12 Muscadinia berry-related traits was performed among 356 individuals from breeding populations of M. rotundifolia. For the transferability of markers between Euvitis and Muscadinia, we used 2000 core genome rhAmpSeq markers developed to allow marker transferability across Euvitis species. A total of 1599 (80%) rhAmpSeq markers returned data in Muscadinia. From the GWAS analyses, we identified a total of 52 quantitative trait nucleotides (QTNs) associated with the 12 berry-related traits. The transferable markers enabled the direct comparison of the QTNs with previously reported results. The whole-genome sequences along with the GWAS results provide a new basis for the extensive study of Muscadinia species.

Identification of a Novel Epigenetic Mechanism of MYC Deregulation in Smoldering and Newly Diagnosed Multiple Myeloma Patients

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM.To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9.To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such as IRF4 and MAF, that play key roles in MM pathogenesis. ChIP-sequencing for these enhancer-associated TFs and luciferase reporter assays targeting their binding sites confirmed the binding and involvement of IRF4 and MAF in activating enhancer elements in MM cells with intact MYC loci.MYC abnormalities are well-known secondary genetic events that trigger the progression of precursor diseases to MM. To define the genetic status of the identified enhancer elements in malignant cells, we examined whole-genome sequencing (WGS) data of 906 MM patients from the MMRF CoMMpass cohort. We found focal amplification of the enhancer region in 2.8% (n = 26) of patients. Transcriptional analysis on the same patient cohort revealed a significant increase in MYC mRNA levels in enhancer-amplified patients compared to MM cases with no MYC aberrations. These results indicate that enhancer activity is required to induce MYC expression and progression of patients with intact MYC loci.Collectively, our findings reveal a novel mechanism of MYC deregulation in malignant plasma cells: selective gain of chromatin accessibility at the enhancer elements and amplification of the enhancer region allow for binding of regulatory factors IRF4 and MAF, which increase the transcription of MYC in the absence of the known MYC chromosomal abnormalities. Our results point to the importance of non-coding regulatory elements and their associated TF networks as drivers of MM progression and suggest a new approach to identify predictive biomarkers and therapeutic targets that could improve patient outcomes in MM and other cancers. DisclosuresFulco: Bristol Myers Squibb: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Chromosome-level genome assembly of the mirid predator Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae), an important natural enemy in the rice ecosystem.

Though the genomes of many rice herbivorous pests have recently been well characterized, little is known about the genome of their natural enemies. Here, by using the Illumina and PacBio platforms, we sequenced and assembled the whole genome of the mirid species Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae), which is an economically and ecologically important natural enemy in the rice ecosystem acting as a dominant predator for planthoppers and leafhoppers in the field. Through Hi-C scaffolding, 1615scaffolds with a total size of 338.08Mb were successfully anchored onto 13 chromosomes. The assembled genome size was 345.75Mb with a final scaffold N50 of 27.58Mb. Approximately 107.51Mb of sequences accounting for 31.10% of the genome were identified as repeat elements, and 14,644 protein-coding genes were annotated. Phylogenetic analysis showed that C.lividipennis clustered with other Hemipteran species and diverged from Apolygus lucorum about 66.7million years ago. Gene families related to detoxification, environmental adaptation and digestion were analysed comparatively with other Hemipteran species, but no significant expansion or contraction was found in C.lividipennis. We also observed male meiosis in C.lividipennis, which showed a typical post-reduction of sex chromosomes and a karyotype of 2n=22+XY. As the first natural-enemy genome in the rice ecosystem, the genomic resource of C.lividipennis not only expands our understanding of the multitrophic interactions (host plant-prey-predator), but also provides a genomic basis for better understanding this dominant predator and therefore promotes sustainable rice pest management and food grain production.

Cotton pan-genome retrieves the lost sequences and genes during domestication and selection

BackgroundMillennia of directional human selection has reshaped the genomic architecture of cultivated cotton relative to wild counterparts, but we have limited understanding of the selective retention and fractionation of genomic components.ResultsWe construct a comprehensive genomic variome based on 1961 cottons and identify 456 Mb and 357 Mb of sequence with domestication and improvement selection signals and 162 loci, 84 of which are novel, including 47 loci associated with 16 agronomic traits. Using pan-genome analyses, we identify 32,569 and 8851 non-reference genes lost from Gossypium hirsutum and Gossypium barbadense reference genomes respectively, of which 38.2% (39,278) and 14.2% (11,359) of genes exhibit presence/absence variation (PAV). We document the landscape of PAV selection accompanied by asymmetric gene gain and loss and identify 124 PAVs linked to favorable fiber quality and yield loci.ConclusionsThis variation repertoire points to genomic divergence during cotton domestication and improvement, which informs the characterization of favorable gene alleles for improved breeding practice using a pan-genome-based approach.

Long-read trio sequencing of individuals with unsolved intellectual disability

Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×–40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

Construction of a chromosome-scale long-read reference genome assembly for potato.

BackgroundWorldwide, the cultivated potato, Solanum tuberosum L., is the No. 1 vegetable crop and a critical food security crop. The genome sequence of DM1–3 516 R44, a doubled monoploid clone of S. tuberosum Group Phureja, was published in 2011 using a whole-genome shotgun sequencing approach with short-read sequence data. Current advanced sequencing technologies now permit generation of near-complete, high-quality chromosome-scale genome assemblies at minimal cost.FindingsHere, we present an updated version of the DM1–3 516 R44 genome sequence (v6.1) using Oxford Nanopore Technologies long reads coupled with proximity-by-ligation scaffolding (Hi-C), yielding a chromosome-scale assembly. The new (v6.1) assembly represents 741.6 Mb of sequence (87.8%) of the estimated 844 Mb genome, of which 741.5 Mb is non-gapped with 731.2 Mb anchored to the 12 chromosomes. Use of Oxford Nanopore Technologies full-length complementary DNA sequencing enabled annotation of 32,917 high-confidence protein-coding genes encoding 44,851 gene models that had a significantly improved representation of conserved orthologs compared with the previous annotation. The new assembly has improved contiguity with a 595-fold increase in N50 contig size, 99% reduction in the number of contigs, a 44-fold increase in N50 scaffold size, and an LTR Assembly Index score of 13.56, placing it in the category of reference genome quality. The improved assembly also permitted annotation of the centromeres via alignment to sequencing reads derived from CENH3 nucleosomes.ConclusionsAccess to advanced sequencing technologies and improved software permitted generation of a high-quality, long-read, chromosome-scale assembly and improved annotation dataset for the reference genotype of potato that will facilitate research aimed at improving agronomic traits and understanding genome evolution.

A genome database for a Japanese population of the larvacean Oikopleura dioica.

The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O.dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O.dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O.dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O.dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.

Genome Improvement and Genetic Map Construction for Aethionema arabicum, the First Divergent Branch in the Brassicaceae Family.

The genus Aethionema is a sister-group to the core-group of the Brassicaceae family that includes Arabidopsis thaliana and the Brassica crops. Thus, Aethionema is phylogenetically well-placed for the investigation and understanding of genome and trait evolution across the family. We aimed to improve the quality of the reference genome draft version of the annual species Aethionema arabicum. Second, we constructed the first Ae. arabicum genetic map. The improved reference genome and genetic map enabled the development of each other. We started with the initially published genome (version 2.5). PacBio and MinION sequencing together with genetic map v2.5 were incorporated to produce the new reference genome v3.0. The improved genome contains 203 MB of sequence, with approximately 94% of the assembly made up of called (non-gap) bases, assembled into 2,883 scaffolds (with only 6% of the genome made up of non-called bases (Ns)). The N50 (10.3 MB) represents an 80-fold increase over the initial genome release. We generated a Recombinant Inbred Line (RIL) population that was derived from two ecotypes: Cyprus and Turkey (the reference genotype. Using a Genotyping by Sequencing (GBS) approach, we generated a high-density genetic map with 749 (v2.5) and then 632 SNPs (v3.0) was generated. The genetic map and reference genome were integrated, thus greatly improving the scaffolding of the reference genome into 11 linkage groups. We show that long-read sequencing data and genetics are complementary, resulting in an improved genome assembly in Ae. arabicum. They will facilitate comparative genetic mapping work for the Brassicaceae family and are also valuable resources to investigate wide range of life history traits in Aethionema.

Analysis of the Yp11.2 Deletion Region of Phenotypically Normal Males with an AMELY-Null Allele in the Chinese Han Population

Aim: The amelogenin gene is a widely used gender marker for forensic DNA profiling. Males who have the amelogenin Y (AMELY) allele deletion can be mistakenly identified as females if genotyping is performed only on the amelogenin gene. The aim of this study was to investigate the frequency of the AMELY allele deletion in the Chinese Han population and analyze the possible genetic variation on the Y chromosome. Materials and Methods: The amelogenin gene of 12,735 unrelated males from the Chinese Han population were genotyped using common forensic short tandem repeat (STR) kits. The AMELY allele deletion was verified by redesigned primers and sequencing. Eighteen Y-specific sequence tagged sites (STSs) on the Yp11.2 region were selected to delineate the deletion breakpoints on the Y chromosome. Results: Three males were confirmed to have no AMELY allele. The frequency rate of the AMELY-null allele was 0.236% (3/12,735) in the Chinese Han population of Central China; 2.73 Mb of sequence on the Y chromosome were absent in all the AMELY-negative samples. The deleted region was mapped using SRY, AMELY, 5 Y-STRs, and 18 STSs, which belong to the class I deleted pattern. The three unrelated males shared the same Y-STR haplotype with four males from other Chinese populations, all of whom have the AMELY-null allele. The haplogroup of these males was identified as the O3 haplogroup. Conclusion: The AMELY allele deletion in the Chinese population was accompanied by the deletion of the Y-STR loci on the Yp11.2 region. Therefore, another Y-specific marker should be tested simultaneously when unknown samples are examined as part of a criminal investigation.

Resolving the full spectrum of human genome variation using Linked-Reads.

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as “Linked-Reads”. This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2. Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

Age-related variation in gene alteration frequency in metastatic prostate cancer.

178 Background: Despite advances in therapy, metastatic prostate cancer (mPCA) remains lethal. Younger men with mPCA have a significantly greater risk of mPCA-related death compared to men diagnosed later in life. This suggests that early-onset mPCA is a uniquely aggressive subtype of disease, however, the genomic drivers have not been explored. Here, we analyze data from 2,196 mPCA specimens profiled as part of routine clinical care. Methods: Comprehensive genomic profiling was performed by hybridization capture of exonic regions from either 236, 315 or 324 cancer-related genes and select introns from 19 genes commonly rearranged in cancer. Libraries were sequenced to high, uniform coverage and assessed for all classes of genomic alterations. Tumor mutational burden (TMB) was determined from 1.1 Mb of sequence and microsatellite instability (MSI) from 114 loci. Results: 336 (15.3%) men were < 60 years old (yo), and 1,860 (84.7%) were ≥60 yo at time of biopsy. Lymph node, liver, and bone were the three most common metastatic sites in both groups, accounting for 75% of all specimens. Patients < 60 yo harbored significantly fewer AR and PTEN point mutations when compared to patients ≥60 yo (OR = 0.48, p < 0.001; OR = 0.51, p = 0.01). Copy number alterations in PTEN and CDKN2A/B were significantly more common among young patients (OR = 1.46, p = 0.003; OR = 1.91, p = 0.04). AR copy number alterations were significantly less common among young patients (OR = 0.57, p < 0.001). Although uncommon in mPCA, markers of genomic instability including MSH2 mutations and MSI were both significantly less common among patients < 60 yo (0.00% vs. 1.29%, p = 0.04; 0.89% vs. 3.06%, p = 0.03). There were no significant differences in the frequency of TP53, APC, BRCA2, ATM, and CDK12 point mutations, MYC copy number alterations, or TMPRSS2-ERG fusions. The average TMB was not significantly different between young and old patients, with 3.26 and 5.79 mutations per Mb, respectively (p = 0.19). Conclusions: Younger men with mPCA appear to represent a genomically distinct disease subtype. To better define this population, we are prospectively investigating the genomic, genetic, and environmental risk factors through an ongoing PCF funded clinical trial.