High-throughput Chromosome Conformation Capture Techniques Research Articles

The Chromosome-Scale Reference Genome of Macadamia tetraphylla Provides Insights Into Fatty Acid Biosynthesis.

Macadamia is an evergreen tree belonging to the Proteaceae family. The two commercial macadamia species, Macadamia integrifolia and M. tetraphylla, are highly prized for their edible kernels. The M. integrifolia genome was recently sequenced, but the genome of M. tetraphylla has to date not been published, which limits the study of biological research and breeding in this species. This study reports a high-quality genome sequence of M. tetraphylla based on the Oxford Nanopore Technologies technology and high-throughput chromosome conformation capture techniques (Hi-C). An assembly of 750.87 Mb with 51.11 Mb N50 length was generated, close to the 740 and 758 Mb size estimates by flow cytometry and k-mer analysis, respectively. Genome annotation indicated that 61.42% of the genome is composed of repetitive sequences and 34.95% is composed of long terminal repeat retrotransposons. Up to 31,571 protein-coding genes were predicted, of which 92.59% were functionally annotated. The average gene length was 6,055 bp. Comparative genome analysis revealed that the gene families associated with defense response, lipid transport, steroid biosynthesis, triglyceride lipase activity, and fatty acid metabolism are expanded in the M. tetraphylla genome. The distribution of fourfold synonymous third-codon transversion showed a recent whole-genome duplication event in M. tetraphylla. Genomic and transcriptomic analysis identified 187 genes encoding 33 crucial oil biosynthesis enzymes, depicting a comprehensive map of macadamia lipid biosynthesis. Besides, the 55 identified WRKY genes exhibited preferential expression in root as compared to that in other tissues. The genome sequence of M. tetraphylla provides novel insights for breeding novel varieties and genetic improvement of agronomic traits.

Whole-genome sequence of Phellinus gilvus (mulberry Sanghuang) reveals its unique medicinal values

Phellinus gilvus (Schwein.) Pat, a species of ‘Sanghuang’, has been well-documented for various medicinal uses, but the genome information and active constituents are largely unknown. Here, we sequenced the whole-genome of P. gilvus, identified phenylpropanoids as its key anti-cancer components, and deduced their biosynthesis pathways. A 41.11-Mb genome sequence was assembled and the heatmap created with high-throughput chromosome conformation capture techniques data suggested all bins could be clearly divided into 11 pseudochromosomes. Cellular experiments showed that P. gilvus fruiting body was more effective to inhibit hepatocellular carcinoma cells than mycelia. High resolution electrospray ionization mass spectroscopy (HR-ESI-MS) analysis revealed P. gilvus fruiting body was rich in phenylpropanoids, and several unique phenylpropanoids in Phellinus spp. exhibited potent anti-carcinogenesis activity. Based on genomic, HR-ESI-MS information and differentially expressed genes in transcriptome analysis, we deduced the biosynthesis pathway of four major phenylpropanoids in P. gilvus. Transcriptome analysis revealed the deduced genes expressions were synergistically changed with the production of phenylpropanoids. The optimal candidate genes of phenylpropanoids’ synthesis pathway were screened by molecular docking analysis. Overall, our results provided a high-quality genomic data of P. gilvus and inferred biosynthesis pathways of four phenylpropanoids with potent anti-carcinogenesis activities. These will be a valuable resource for further genetic improvement and effective use of the P. gilvus.

A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine

Isatis indigotica (2n = 14) is an important medicinal plant in China. Its dried leaves and roots (called Isatidis Folium and Isatidis Radix, respectively) are broadly used in traditional Chinese medicine for curing diseases caused by bacteria and viruses such as influenza and viral pneumonia. Various classes of compounds isolated from this species have been identified as effective ingredients. Previous studies based on transcriptomes revealed only a few candidate genes for the biosynthesis of these active compounds in this medicinal plant. Here, we report a high-quality chromosome-scale genome assembly of I. indigotica with a total size of 293.88 Mb and scaffold N50 = 36.16 Mb using single-molecule real-time long reads and high-throughput chromosome conformation capture techniques. We annotated 30,323 high-confidence protein-coding genes. Based on homolog searching and functional annotations, we identified many candidate genes involved in the biosynthesis of main active components such as indoles, terpenoids, and phenylpropanoids. In addition, we found that some key enzyme-coding gene families related to the biosynthesis of these components were expanded due to tandem duplications, which likely drove the production of these major active compounds and explained why I. indigotica has excellent antibacterial and antiviral activities. Our results highlighted the importance of genome sequencing in identifying candidate genes for metabolite synthesis in medicinal plants.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for ‘Youshi’ (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembled D. oleifera genome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that the D. oleifera genome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO2 treatment to remove astringency. Using the D. oleifera genome as the reference, SLAF-seq indicated that ‘Youshi’ is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.

Whole-genome sequencing and analysis of the Chinese herbal plant Gelsemium elegans

BackgroundGelsemium elegans (G. elegans) (2n = 2x = 16) is genus of flowering plants belonging to the Gelsemicaeae family. MethodHere, a high-quality genome assembly using the Oxford Nanopore Technologies (ONT) platform and high-throughput chromosome conformation capture techniques (Hi-C) were used. ResultsA total of 56.11 Gb of raw GridION X5 platform ONT reads (6.23 Gb per cell) were generated. After filtering, 53.45 Gb of clean reads were obtained, giving 160 × coverage depth. The de novo genome assemblies 335.13 Mb, close to the 338 Mb estimated by k-mer analysis, was generated with contig N50 of 10.23 Mb. The vast majority (99.2%) of the G. elegans assembled sequence was anchored onto 8 pseudo-chromosomes. The genome completeness was then evaluated and 1338 of the 1440 conserved genes (92.9%) could be found in the assembly. Genome annotation revealed that 43.16% of the G. elegans genome is composed of repetitive elements and 23.9% is composed of long terminal repeat elements. We predicted 26,768 protein-coding genes, of which 84.56% were functionally annotated. ConclusionThe genomic sequences of G. elegans could be a valuable source for comparative genomic analysis in the Gelsemicaeae family and will be useful for understanding the phylogenetic relationships of the indole alkaloid metabolism.

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1–4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3–79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

Promoter capture Hi-C-based identification of recurrent noncoding mutations in colorectal cancer

Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations1-6. cis-regulatory elements (CREs) represent a highly enriched subset of the non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas7,8. We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification1. This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.

Identification of copy number variations and translocations in cancer cells from Hi-C data.

Eukaryotic chromosomes adapt a complex and highly dynamic three-dimensional (3D) structure, which profoundly affects different cellular functions and outcomes including changes in epigenetic landscape and in gene expression. Making the scenario even more complex, cancer cells harbor chromosomal abnormalities [e.g. copy number variations (CNVs) and translocations] altering their genomes both at the sequence level and at the level of 3D organization. High-throughput chromosome conformation capture techniques (e.g. Hi-C), which are originally developed for decoding the 3D structure of the chromatin, provide a great opportunity to simultaneously identify the locations of genomic rearrangements and to investigate the 3D genome organization in cancer cells. Even though Hi-C data has been used for validating known rearrangements, computational methods that can distinguish rearrangement signals from the inherent biases of Hi-C data and from the actual 3D conformation of chromatin, and can precisely detect rearrangement locations de novo have been missing. In this work, we characterize how intra and inter-chromosomal Hi-C contacts are distributed for normal and rearranged chromosomes to devise a new set of algorithms (i) to identify genomic segments that correspond to CNV regions such as amplifications and deletions (HiCnv), (ii) to call inter-chromosomal translocations and their boundaries (HiCtrans) from Hi-C experiments and (iii) to simulate Hi-C data from genomes with desired rearrangements and abnormalities (AveSim) in order to select optimal parameters for and to benchmark the accuracy of our methods. Our results on 10 different cancer cell lines with Hi-C data show that we identify a total number of 105 amplifications and 45 deletions together with 90 translocations, whereas we identify virtually no such events for two karyotypically normal cell lines. Our CNV predictions correlate very well with whole genome sequencing data among chromosomes with CNV events for a breast cancer cell line (r = 0.89) and capture most of the CNVs we simulate using Avesim. For HiCtrans predictions, we report evidence from the literature for 30 out of 90 translocations for eight of our cancer cell lines. Furthermore, we show that our tools identify and correctly classify relatively understudied rearrangements such as double minutes and homogeneously staining regions. Considering the inherent limitations of existing techniques for karyotyping (i.e. missing balanced rearrangements and those near repetitive regions), the accurate identification of CNVs and translocations in a cost-effective and high-throughput setting is still a challenge. Our results show that the set of tools we develop effectively utilize moderately sequenced Hi-C libraries (100-300 million reads) to identify known and de novo chromosomal rearrangements/abnormalities in well-established cancer cell lines. With the decrease in required number of cells and the increase in attainable resolution, we believe that our framework will pave the way towards comprehensive mapping of genomic rearrangements in primary cells from cancer patients using Hi-C. CNV calling: https://github.com/ay-lab/HiCnv, Translocation calling: https://github.com/ay-lab/HiCtrans and Hi-C simulation: https://github.com/ay-lab/AveSim. Supplementary data are available at Bioinformatics online.