Non-coding Research Articles

Analysis of the complete mitochondrial genome sequence of Hipposideros pratti

In order to explore the characteristics of the mitochondrial genome sequence of Pratt’s leaf-nosed bat (Hipposideros pratti Thomas 1891) and understand their phylogenetic status in Chiroptera, this study determined the mitochondrial genome sequences of H. pratti from five regions in China using high-throughput sequencing technology, sequence assembly, and genome annotation. The results showed that these sequences contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 non-coding region, all exhibiting a significant AT bias. Based on the phylogenetic tree constructed using 13 protein-coding genes from 15 Chiroptera species, the study found that H. pratti from the five regions clustered together, and then clustered with H. lylei into a single clade. Meanwhile, H. pratti from Jiangxi, Fujian, and Guangdong regions of China showed closer genetic relationships, while H. pratti from Yunnan and Henan regions of China exhibited closer genetic relationships. This study not only supplemented the mitochondrial genome database of H. pratti but also laid a foundation for genetic variation, molecular classification, and evolutionary studies of H. pratti.

Machine learning-based classification reveals distinct clusters of non-coding genomic allelic variations associated with Erm-mediated antibiotic resistance.

Antibiotic resistance (AR) poses a global health threat as the efficacy of available antibiotics has rapidly eroded due to the widespread transmission of AR genes. Using Erm-dependent MLS resistance as a model, this study highlights the significance of non-coding genomic allelic variations. Through a comprehensive analysis of upstream regulatory elements within the erm family, we elucidated the evolutionary emergence and development of AR mechanisms. Leveraging population-wide machine learning (ML)-based genomic analysis, we transformed substantial non-random allelic variations into discernible clusters of elements, enabling precise prediction of MLS phenotypes from non-coding regions. These findings offer deeper insight into AR evolution and demonstrate the potential of harnessing non-coding genomic allele data for accurately predicting AR phenotypes.

Phylogenomic analysis and dynamic evolution of chloroplast genomes of Clematis nannophylla

Clematis nannophylla is a perennial shrub of Clematis with ecological, ornamental, and medicinal value, distributed in the arid and semi-arid areas of northwest China. This study successfully determined the chloroplast (cp) genome of C. nannophylla, reconstructing a phylogenetic tree of Clematis. This cp genome is 159,801 bp in length and has a typical tetrad structure, including a large single-copy, a small single-copy, and a pair of reverse repeats (IRa and IRb). It contains 133 unique genes, including 89 protein-coding, 36 tRNA, and 8 rRNA genes. Additionally, 66 simple repeat sequences, 50 dispersed repeats, and 24 tandem repeats were found; many of the dispersed and tandem repeats were between 20–30 bp and 10–20 bp, respectively, and the abundant repeats were located in the large single copy region. The cp genome was relatively conserved, especially in the IR region, where no inversion or rearrangement was observed, further revealing that the coding regions were more conserved than the noncoding regions. Phylogenetic analysis showed that C. nannophylla is more closely related to C. fruticosa and C. songorica. Our analysis provides reference data for molecular marker development, phylogenetic analysis, population studies, and cp genome processes to better utilise C. nannophylla.

Identification and validation of tumor-specific T cell receptors from tumor infiltrating lymphocytes using tumor organoid co-cultures

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy.

Machine learning enables pan-cancer identification of mutational hotspots at persistent CTCF binding sites.

CCCTC-binding factor (CTCF) is an insulator protein that binds to a highly conserved DNA motif and facilitates regulation of three-dimensional (3D) nuclear architecture and transcription. CTCF binding sites (CTCF-BSs) reside in non-coding DNA and are frequently mutated in cancer. Our previous study identified a small subclass of CTCF-BSs that are resistant to CTCF knock down, termed persistent CTCF binding sites (P-CTCF-BSs). P-CTCF-BSs show high binding conservation and potentially regulate cell-type constitutive 3D chromatin architecture. Here, using ICGC sequencing data we made the striking observation that P-CTCF-BSs display a highly elevated mutation rate in breast and prostate cancer when compared to all CTCF-BSs. To address whether P-CTCF-BS mutations are also enriched in other cell-types, we developed CTCF-INSITE-a tool utilising machine learning to predict persistence based on genetic and epigenetic features of experimentally-determined P-CTCF-BSs. Notably, predicted P-CTCF-BSs also show a significantly elevated mutational burden in all 12 cancer-types tested. Enrichment was even stronger for P-CTCF-BS mutations with predicted functional impact to CTCF binding and chromatin looping. Using in vitro binding assays we validated that P-CTCF-BS cancer mutations, predicted to be disruptive, indeed reduced CTCF binding. Together this study reveals a new subclass of cancer specific CTCF-BS DNA mutations and provides insights into their importance in genome organization in a pan-cancer setting.

The complete mitochondrial genome and phylogenetic analysis of Pealius mori (Hemiptera: Aleyrodidae)

The complete mitochondrial genome of Pealius mori (Hemiptera: Aleyrodidae) was determined in this study. The mitogenome was 15,654 bp long with 37 typical Insecta mitochondrial genes and one non-coding control region. Its gene content and order were different to other Hemiptera mitochondrial genomes. The overall nucleotide composition of the mitogenome was 42.62% A, 32.73% T, 11.12% G and 13.54% C, with an A + T bias of 75.34%. Phylogenetic analyses of 14 species in Aleyrodidae, 2 species in Lepidoptera and 1 species in Thysanoptera by Maximum Likelihood showed that P. mori China had been more closely related to P. mori France, closely related to Pealius machili. This result well supported the taxonomic position of Aleyrodidae and their close relationship with the Pealius category.

A-to-I Editing Is Subtype-Specific in Non-Hodgkin Lymphomas.

Cancer is a complex and heterogeneous disease, in which a number of genetic and epigenetic changes occur in tumor onset and progression. Recent studies indicate that changes at the RNA level are also involved in tumorigenesis, such as adenosine-to-inosine (A-to-I) RNA editing. Here, we systematically investigate transcriptome-wide A-to-I editing events in a large number of samples from Non-Hodgkin lymphomas (NHLs). Using a computational pipeline that determines significant differences in editing level between NHL and normal samples at known A-to-I editing sites, we identify a number of differentially edited editing sites between NHL subtypes and normal samples. Most of the differentially edited sites are located in non-coding regions, and many such sites show a strong correlation between gene expression level and editing efficiency, indicating that RNA editing might have direct consequences for the cancer cell's aberrant gene regulation status in these cases. Moreover, we establish a strong link between RNA editing and NHL by demonstrating that NHL and normal samples and even NHL subtypes can be distinguished based on genome-wide RNA editing profiles alone. Our study establishes a strong link between RNA editing, cancer and aberrant gene regulation in NHL.

CapsEnhancer: An Effective Computational Framework for Identifying Enhancers Based on Chaos Game Representation and Capsule Network.

Enhancers are a class of noncoding DNA, serving as crucial regulatory elements in governing gene expression by binding to transcription factors. The identification of enhancers holds paramount importance in the field of biology. However, traditional experimental methods for enhancer identification demand substantial human and material resources. Consequently, there is a growing interest in employing computational methods for enhancer prediction. In this study, we propose a two-stage framework based on deep learning, termed CapsEnhancer, for the identification of enhancers and their strengths. CapsEnhancer utilizes chaos game representation to encode DNA sequences into unique images and employs a capsule network to extract local and global features from sequence "images". Experimental results demonstrate that CapsEnhancer achieves state-of-the-art performance in both stages. In the first and second stages, the accuracy surpasses the previous best methods by 8 and 3.5%, reaching accuracies of 94.5 and 95%, respectively. Notably, this study represents the pioneering application of computer vision methods to enhancer identification tasks. Our work not only contributes novel insights to enhancer identification but also provides a fresh perspective for other biological sequence analysis tasks.

In silico generation and augmentation of regulatory variants from massively parallel reporter assay using conditional variational autoencoder.

Predicting the functional consequences of genetic variants in non-coding regions is a challenging problem. Massively parallel reporter assays (MPRAs), which are an in vitro high-throughput method, can simultaneously test thousands of variants by evaluating the existence of allele specific regulatory activity. Nevertheless, the identified labelled variants by MPRAs, which shows differential allelic regulatory effects on the gene expression are usually limited to the scale of hundreds, limiting their potential to be used as the training set for achieving a robust genome-wide prediction. To address the limitation, we propose a deep generative model, MpraVAE, to in silico generate and augment the training sample size of labelled variants. By benchmarking on several MPRA datasets, we demonstrate that MpraVAE significantly improves the prediction performance for MPRA regulatory variants compared to the baseline method, conventional data augmentation approaches as well as existing variant scoring methods. Taking autoimmune diseases as one example, we apply MpraVAE to perform a genome-wide prediction of regulatory variants and find that predicted regulatory variants are more enriched than background variants in enhancers, active histone marks, open chromatin regions in immune-related cell types, and chromatin states associated with promoter, enhancer activity and binding sites of cMyC and Pol II that regulate gene expression. Importantly, predicted regulatory variants are found to link immune-related genes by leveraging chromatin loop and accessible chromatin, demonstrating the importance of MpraVAE in genetic and gene discovery for complex traits.

DNA Transposons Favor De Novo Transcript Emergence Through Enrichment of Transcription Factor Binding Motifs.

De novo genes emerge from noncoding regions of genomes via succession of mutations. Among others, such mutations activate transcription and create a new open reading frame (ORF). Although the mechanisms underlying ORF emergence are well documented, relatively little is known about the mechanisms enabling new transcription events. Yet, in many species a continuum between absent and very prominent transcription has been reported for essentially all regions of the genome. In this study, we searched for de novo transcripts by using newly assembled genomes and transcriptomes of seven inbred lines of Drosophila melanogaster, originating from six European and one African population. This setup allowed us to detect sample specific de novo transcripts, and compare them to their homologous nontranscribed regions in other samples, as well as genic and intergenic control sequences. We studied the association with transposable elements (TEs) and the enrichment of transcription factor motifs upstream of de novo emerged transcripts and compared them with regulatory elements. We found that de novo transcripts overlap with TEs more often than expected by chance. The emergence of new transcripts correlates with regions of high guanine-cytosine content and TE expression. Moreover, upstream regions of de novo transcripts are highly enriched with regulatory motifs. Such motifs are more enriched in new transcripts overlapping with TEs, particularly DNA TEs, and are more conserved upstream de novo transcripts than upstream their 'nontranscribed homologs'. Overall, our study demonstrates that TE insertion is important for transcript emergence, partly by introducing new regulatory motifs from DNA TE families.

A Potential Pathogenic SRD5A2 Mutation and rs632148, rs523349 and rs522638 Polymorphisms in Increasing the Risk of Syndromic Hypospadias in Indonesian Population

BACKGROUND: Hypospadias, a congenital birth defect in male, is the opening of the urethra located on the ventral side of the penis. Several mutations in SRD5A2 encoding steroid 5 alpha-reductase type 2 protein have been identified in hypospadias and polymorphisms in this gene have been known to be associated with an increased risk of hypospadias. In this study, several crucial molecular analyses of the SRD5A2 gene and the association of the identified variants to the risk of syndromic hypospadias in Indonesian population were conducted.METHODS: Thirty-two isolated and 29 syndromic hypospadias patients were enrolled in this study. DNA was isolated from whole blood for the amplification of all exons and exon-intron boundaries of SRD5A2 by polymerase chain reaction (PCR), followed by Sanger sequencing. In silico analysis was performed using PolyPhen-2, Sorting Intolerant from Tolerant (SIFT) and Align GVGD. Statistical analysis was performed using Chi-squared test.RESULTS: A novel missense mutation c.32T>C/p.Leu11Pro was identified in one isolated hypospadias patient and the in silico analysis predicted the mutation to be pathogenic. Three polymorphisms were identified, two in the non-coding region (c.-62G>C/rs632148 and c.281+15T>C/rs522638) and one in exon-1 (c.265C>G/p.Val89Leu/rs523349). Mutant alleles of these polymorphisms were significantly associated with syndromic hypospadias with odds ratios (OR) of 3.4, 3.13 and 2.54 respectively.CONCLUSION: This study suggests that SRD5A2 mutation is one of the causes of hypospadias in Indonesian population and rs632148, rs523349 and rs522638 polymorphisms are significantly associated with an increased risk of syndromic hypospadias.KEYWORDS: mutation, polymorphism, SRD5A2, syndromic hypospadias

Mitochondrial genome analysis reveals phylogenetic insights and gene rearrangements in Parupeneus (Syngnathiformes: Mullidae)

Despite the critical role of mitochondrial genomes (mitogenomes) in species identification and evolutionary studies in the genus Parupeneus, current resources are inadequate, given the species richness. Although previous studies have suggested a complex evolutionary history, the detailed mitogenomic variations and their implications remain largely unexplored. Therefore, we sequenced and assembled the mitogenomes of P. barberinoides, P. barberinus, P. biaculeatus, P. crassilabris, P. cyclostomus, P. heptacanthus, P. multifasciatus, and P. chrysopleuron, to enrich the molecular data and provide novel insights into the genetic diversity, evolutionary dynamics and phylogenetics of the family Mullidae. Our analysis revealed a novel gene rearrangement in P. chrysopleuron, Cytb-T-P-CR-Q-I-F-12S-V-16S-ND1-M-ND2, which differed from the conventional sequence of Cytb-T-P-CR-F-12S-V-16S-ND1-I-Q-M-ND2 observed in other species. In the novel rearrangement, four non-coding regions are inserted between ND1 and M, Q and I, I and ψM (tRNA-Met pseudogene), ψM and F. We assume that two tandem duplication/random loss events occur in the CR and IQM, making the entire sequence longer than that in other Parupeneus species. The phylogenetic results indicated that Mullidae formed a sister group relationship with the family Dactylopteridae, contradicting previous studies that identified a sister group relationship between Mullidae and Callionymoidei. The genera Parupeneus and Mullus formed a sister group, and discrepancies were found in the topological structure of the interspecies relationships within the genus Parupeneus compared with those reported by previous studies. Through combined phylogenetic and mitochondrial structural analysis, we found that phylogenetic topology is closely related to mitochondrial structural abnormalities. This study not only expands the mitogenomic dataset available for Mullidae but also underscores the importance of mitochondrial DNA studies in resolving taxonomic ambiguities and understanding the evolutionary history of marine fishes. Our study contributes to the ongoing research on marine fish taxonomy, mitogenomics, and evolutionary biology by providing new insights into the genetic diversity of marine ecosystems.

Acquired copy number amplification at the MYC enhancer in human B-precursor acute lymphoblastic leukemia cell lines.

Our study highlights the discovery of recurrent copy number alterations in noncoding regions, specifically blood enhancer cluster (BENC-CNA), in B-precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. We demonstrate that BENC-CNA acts as a super-enhancer, driving MYC expression and possibly contributing to the immortalization and proliferative advantage of BCP-ALL cells invitro.

ProtoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQRand enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

Unraveling the complex evolutionary features of the Cinnamomum camphora mitochondrial genome.

We reported the mitochondrial genome of Cinnamomum camphora for the first time, revealing frequent rearrangement events in the non-coding regions of Magnoliids mitochondrial genomes. As one of the representative species in the Lauraceae family of Magnoliids, Cinnamomum camphora holds significant economic and ecological value. In this study, the mitochondrial genome (mitogenome) of C. camphora was complete assembled and annotated using PacBio HiFi sequencing. The C. camphora mitogenome is characterized by a branch structure, spans 900,894 bp, and contains 43 protein-coding genes (PCGs), 24 tRNAs, and 3 rRNAs. Most of these PCGs are under purifying selection, with only two (ccmFc and rps7) exhibiting signs of positive selection. The C. camphora mitogenome contains numerous repetitive sequences and intracellular gene transfers, with a total of 36 mitochondrial plastid DNAs, amounting to a combined length of 23,816 bp. Comparative analysis revealed that the non-coding regions of Magnoliids mitogenomes have undergone frequent rearrangements during evolution, but the coding sequences remain highly conserved (more than 98% similarity for protein-coding sequences). Furthermore, a maximum-likelihood phylogenetic tree was reconstructed based on 25 PCGs from 23 plant mitogenomes. The analysis supports the closest relationship between C. camphora and C. chekiangense, consistent with the APG IV classification system. This study elucidates the unique evolutionary features of the C. camphora mitogenome, which will provide valuable insights into the study of genetics and evolution of the family Lauraceae.

Complete chloroplast genomes of eight Delphinium taxa (Ranunculaceae) endemic to Xinjiang, China: insights into genome structure, comparative analysis, and phylogenetic relationships

BackgroundDelphinium L. represents a taxonomically intricate genus of significant phylogenetic and economic importance in Ranunculaceae. Despite the existence of few chloroplast genome datasets, a comprehensive understanding of genome structures and selective pressures within the genus remains unknown. Furthermore, several taxa in this genus are exclusively found in Xinjiang, China, a region renowned for its distribution and diversity of Chinese and Central Asian Delphinium species. Therefore, investigating the features of chloroplast genomes in this area will provide valuable insights into the evolutionary processes and phylogenetic relationships of the genus.ResultsIn this study, the eight newly completed chloroplast genomes are examined, ranging in length from 153,979 bp to 154,284 bp. Alongside these, analysing six previously reported taxa re-annotated in Delphinium, 111 unique genes are identified across all samples. Genome structure, distributions of simple sequence repeats and short dispersed repeats, as well as gene content are similar among these Delphinium taxa. Nine hypervariable intergenic spacers and protein coding regions, including ndhF-trnL(TAG), rpl16-intron, rpl33, rps15, rps18, trnK(TTT)-trnQ(TTG), trnP(TGG)-psaJ, trnT(GGT)-psbD and ycf1, are identified among 13 perennial Delphinium. Selective pressure and codon usage bias of all the plastid genes are performed within 14 Delphinium taxa. Phylogenetic analysis based on 14 Delphinium plastomes, alongside two Aconitum (Ranunculaceae) species serving as outgroup taxa, reveals the monophyletic nature of Delphinium. Our findings further discern Delphinium into two distinct clades: perennial species (clade I) and annual species (clade II). In addition, compared with the nrDNA ITS topology, cytological data and morphological characters, D. mollifolium and D. maackianum showed potential involvement in hybridization or polyploidization processes. Excluding these two species, the perennial Delphinium (clade I) exhibits a stronger consistency with the morphology-based system that utilized seed morphology.ConclusionThis study represents the first comprehensive analysis of plastomic variations among Delphinium taxa, based on the examination of 14 complete plastomes. The chloroplast genome structure of Delphinium is similar to other angiosperms and possesses the typical quadripartite structure with the conserved genome arrangement and gene features. In addition, the variation of non-coding regions is larger than coding regions of the chloroplast genome. Through DNA sequence divergence across Delphinium plastomes and subsequent phylogenomic analyses ndhF-trnL(TAG) and ycf1 are identified as promising molecular markers. These highly variable loci held significant potential for future phylogenetic and phylogeographic studies on Delphinium. Our phylogenomic analyses based on the whole plastomes, concatenation of 132 unique intergenic spacer regions, concatenation of 77 unique protein-coding genes and nrDNA ITS, all support the monophyly of Delphinium and perennial taxa clusters together into one clade within this genus. These findings provide crucial data for systematic, phylogenomic and evolutionary research in the genus for future studies.

Emergence of a novel GIII Getah virus variant in pigs in Guangdong, China, 2023.

From May to July of 2023, one pig farm in Heyuan city, Guangdong Province of China, suffered severe piglet death and sow reproductive disorders. The common pig viruses and bacteria tested negative. To uncover the possible cause of the disease, a metagenomic analysis was performed in the pooled small intestine samples from three 8-day-old diseased piglets. The results showed that Getah virus (GETV), an RNA virus, might be the potential pathogen that affects pig health. Subsequently, GETV nucleotide was detected in all of the 15 samples collected from three diseased piglets using quantitative reverse transcription PCR, suggesting GETV as the main pathogen of the disease. A GETV strain, designated as GDHYLC23, was successfully isolated using the swine testicle cell line. Sequence analysis showed that the epidemic strain had a unique 32-nucleotide repeat insertion in the 3' noncoding region. Phylogenetic analysis showed that GDHYLC23 belonged to the pandemic group III. The identification of GETV with new variations implies the continuous evolution of the virus, which poses potential threats to the swine industry.IMPORTANCEPig farms are faced with emerging and re-emerging viruses that may cause substantial economic loss. The identification of potentially pathogenic viruses helps to prevent and control the spread of diseases. In this study, by using metagenomic analysis, we found that a neglected virus, GETV with a unique insertion in the genome, was the main pathogen in one pig farm that suffered severe piglet death and sow reproductive disorders. Although the potential impact of such an insertion on viral pathogenicity is unknown, the surveillance of the continuing evolution of GETV in pig farms cannot be ignored.

Revealing the complete mtDNA genome sequence of Cemani chicken (Gallus gallus) by using Nanopore sequencing analysis.

This study aimed to identify, discover and explore the characteristics of the mtDNA genomes of Cemani chicken (Gallus gallus). This study used gDNA of Cemani chicken isolated from liver tissue. mtDNA sequencing was performed using WGS mtDNA analysis with nanopore technology by Oxford Nanopore Technologies GridION. Bioinformatics and data analysis were then performed. This study showed that the length of the mtDNA genome is 16,789 bp, consisting of two ribosomal RNA (12S rRNA, 16S rRNA), 22 transfer RNA genes (trnR, trnG, trnK, trnD, trnS, trnY, trnC, trnN, trnA, trnW, trnM, trnQ, trnl, trnL, trnV, trnF, trnP, trnT, trnE, trnL, trnS, trnH), 13 protein-coding genes (PCGs) (ND4l, ND3, COX3, ATP6, ATP8, COX2, COX1, ND2, ND1, CYTB, ND6, ND5, ND4), and a noncoding control region (Dloop). Furthermore, analysis showed there were polymorphic sites and amino acid alterations when mtDNA Cemani chicken was aligned with references from GenBank. Site (988T>*) in Dloop genes and (328A>G) in ND3 genes which alter glycine to stop codon, were specific markers found only in Cemani chicken.

Complete Mitochondrial Genome and Phylogenetic Analysis of the Blue Whistling Thrush (Myophonus caeruleus).

The blue whistling thrush (Myophonus caeruleus) is a bird belonging to the order Passeriformes and family Muscicapidae. M. caeruleus is widely distributed in China, Pakistan, India, and Myanmar and is a resident bird in the southern part of the Yangtze River in China and summer migratory bird in the northern part of the Yangtze River. At present, there are some controversies about the classification of M. caeruleus. We use complete mitochondrial genomes to provide insights into the phylogenetic position of M. caeruleus and its relationships among Muscicapidae. The mitochondrial genome (GenBank: MN564936) is 16,815 bp long and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding control region (D-loop). The thirteen PCGs started with GTG and ATG and ended with five types of stop codons. The nucleotide composition of T was 23.71%, that of C was 31.45%, that of A was 30.06%, and that of G was 14.78%. The secondary structures of 22 tRNAs were predicted, all of which could form typical cloverleaf structures. There were 24 mismatches, mainly G-U mismatches. Through phylogenetic tree reconstruction, it was found that Saxicola, Monticola, Oenanthe, and Phoenicurus were clustered into one clade, together with the sister group of Myophonus.

Differential impact of quiescent non-coding loci on chromatin entropy.

Non-coding regions of the human genome are important for functional regulations, but their mechanisms remain elusive. We used machine learning to guide a CRISPR screening on hubs (i.e. non-coding loci forming many 3D contacts) and significantly increased the discovery rate of hubs essential for cell growth. We found no clear genetic or epigenetic differences between essential and nonessential hubs, but we observed that some neighboring hubs in the linear genome have distinct spatial contacts and opposite effects on cell growth. One such pair in an epigenetically quiescent region showed different impacts on gene expression, chromatin accessibility and chromatin organization. We also found that deleting the essential hub altered the genetic network activity and increased the entropy of chromatin accessibility, more severe than that caused by deletion of the nonessential hub, suggesting that they are critical for maintaining an ordered chromatin structure. Our study reveals new insights into the system-level roles of non-coding regions in the human genome.