Abstract The successful accomplishment of the first telomere-to-telomere human genome assembly, T2T-CHM13, marked a milestone in achieving completeness of the human reference genome. The upcoming era of genome study will focus on fully phased diploid genome assembly, with an emphasis on genetic differences between individual haplotypes. Most existing sequencing approaches only achieved localized haplotype phasing and relied on additional pedigree information for further whole-chromosome scale phasing. The short-read-based Strand-seq method is able to directly phase single nucleotide polymorphisms (SNPs) at whole-chromosome scale but falls short when it comes to phasing structural variations (SVs). To shed light on this issue, we developed a Nanopore sequencing platform-based Strand-seq approach, which we named NanoStrand-seq. This method allowed for de novo SNP calling with high precision (99.52%) and acheived a superior phasing accuracy (0.02% Hamming error rate) at whole-chromosome scale, a level of performance comparable to Strand-seq for haplotype phasing of the GM12878 genome. Importantly, we demonstrated that NanoStrand-seq can efficiently resolve the MHC locus, a highly polymorphic genomic region. Moreover, NanoStrand-seq enabled independent direct calling and phasing of deletions and insertions at whole-chromosome level; when applied to long genomic regions of SNP homozygosity, it outperformed the strategy that combined Strand-seq with bulk long-read sequencing. Finally, we showed that, like Strand-seq, NanoStrand-seq was also applicable to primary cultured cells. Together, here we provided a novel methodology that enabled interrogation of a full spectrum of haplotype-resolved SNPs and SVs at whole-chromosome scale, with broad applications for species with diploid or even potentially polypoid genomes.
Objective To investigate the prevalence of depression or anxiety in patient with multiple myeloma (MM) in China during maintenance treatment and its associated influencing factors. Methods Patients with MM ( n = 160) received maintenance therapy, and control subjects (without MM, n = 160) matched on age, sex, and BMI were recruited. Patients completed questionnaires, including the Patient Health Questionnaire-9 (PHQ-9), the Generalized Anxiety Disorder 7-item Scale (GAD-7), and the Verbal Pain Rating Scale (VPRS). Data on the Clinical characteristics, biochemical indicators of de novo MM were from the database of the Hematology Department of Beijing Chao-yang Hospital. Multiple linear regression model analysis was used to compare the differences in PHQ-9 and GAD-7 scale scores between the control group and the case group after correction for relevant variables. Multiple logistic regression models were subsequently used to analyze the correlation between the presence or absence of anxiety and depression and clinical indicators in the MM groups. Results Depression symptoms was present in 33.33% and anxiety symptoms in 24.68% of first-episode MM in the maintenance phase of treatment, and depression symptoms in the index-corrected MM group was significantly different from that in the control group ( t = 2.54, P < 0.05). Analyses of multiple logistic regressions: biochemical indicators and clinical typing were not significantly associated with anxiety and depression. Compared to the pain rating 1, the risk of depressive mood was greater in the case group with the pain rating 2 (OR = 2.38) and the pain rating ≥ 3 (OR = 4.32). The risk of anxiety was greater in the case group with the pain rating ≥ 3 than the pain rating 1 (OR = 2.89). Conclusion Despite being in clinical remission, depressive mood problems in patients with MM remain prominent. Clinicians should enhance mood assessment and management in patients with concomitant pain.
Abstract The anterior pituitary gland plays a central role in regulating various physiological processes, including body growth, reproduction, metabolism and stress response. Here, we perform single-cell RNA-sequencing (scRNA-seq) of 4113 individual cells from human fetal pituitaries. We characterize divergent developmental trajectories with distinct transitional intermediate states in five hormone-producing cell lineages. Corticotropes exhibit an early intermediate state prior to full differentiation. Three cell types of the PIT-1 lineage (somatotropes, lactotropes and thyrotropes) segregate from a common progenitor coexpressing lineage-specific transcription factors of different sublineages. Gonadotropes experience two multistep developmental trajectories. Furthermore, we identify a fetal gonadotrope cell subtype expressing the primate-specific hormone chorionic gonadotropin. We also characterize the cellular heterogeneity of pituitary stem cells and identify a hybrid epithelial/mesenchymal state and an early-to-late state transition. Here, our results provide insights into the transcriptional landscape of human pituitary development, defining distinct cell substates and subtypes and illustrating transcription factor dynamics during cell fate commitment.
Abstract Colorectal cancer is a highly heterogeneous disease, with well-characterized subtypes based on genome, DNA methylome, and transcriptome signatures. To chart the epigenetic landscape of colorectal cancers, we generated a high-quality single-cell chromatin accessibility atlas of epithelial cells for 29 patients. Abnormal chromatin states acquired in adenomas were largely retained in colorectal cancers, which were tightly accompanied by opposite changes of DNA methylation. Unsupervised analysis on malignant cells revealed two epigenetic subtypes, exactly matching the iCMS classification, and key iCMS-specific transcription factors (TFs) were identified, including HNF4A and PPARA for iCMS2 tumors and FOXA3 and MAFK for iCMS3 tumors. Notably, subtype-specific TFs bind to distinct target gene sets and contribute to both interpatient similarities and diversities for both chromatin accessibilities and RNA expressions. Moreover, we identified CpG-island methylator phenotypes and pinpointed chromatin state signatures and TF regulators for the CIMP-high subtype. Our work systematically revealed the epigenetic basis of the well-known iCMS and CIMP classifications of colorectal cancers. Significance: Our work revealed the epigenetic basis of the well-known iCMS and CIMP classifications of colorectal cancers. Moreover, interpatient minor similarities and major diversities of chromatin accessibility signatures of TF target genes can faithfully explain the corresponding interpatient minor similarities and major diversities of RNA expression signatures of colorectal cancers, respectively. This article is featured in Selected Articles from This Issue, p. 897
AML with Mt NPM1 has relatively good responses to induction therapy. However, a proportion of NPMc+ AML cells cannot be cleared by conventional treatments. Therefore, we determined the therapeutic efficacy of deguelin that has demonstrated extensive biological activity with low toxicity. We previously reported that deguelin selectively reduces Mt NPM1, as well as induces differentiation and potentiates apoptosis in NPMc+ AML cells. Nevertheless, little information is available regarding the mechanism of deguelin-induced differentiation. Here, we investigated the role of deguelin in the induction of NPMc+ AML cell differentiation. Deguelin at the nontoxic concentration of 2 μM strongly inhibited cell growth but reduced apoptosis in OCI-AML3 cells carrying Mt NPM1, whereas the antiproliferative effect was minimal in OCIM2 cells harboring Wt NPM1. Compared with OCIM2 cells that showed no response, deguelintreated OCI-AML3 cells exhibited the morphological features of granulocytic/monocytic differentiation, increased expression of differentiation antigens, and a nitroblue tetrazolium reduction activity. Induction of differentiation was associated with downregulation of Mt NPM1 and SIRT1, but not Wt NPM1, which was accompanied by an increase in CEBPβ and G-CSFR expression, and further confirmed by sh-Mt NPM1 and sh-SIRT1. sh-Mt NPM1 treatment reduced SIRT1 expression, but did not change HDAC1/3 levels, suggesting that the decline of SIRT1 was partially accountable for the deguelin-induced, Mt-NPM1-related differentiation. Moreover, Mt NPM1 overexpression blocked deguelin-induced cell differentiation. Lastly, we showed that deguelin reduced the expression of Mt NPM1 via the ubiquitin-proteasome pathway. Taken together, our results suggest that deguelin may be a therapeutic candidate for NPMc+ AML. Keywords: Deguelin differentiation NPMc+ AML.
Although localized haploid phasing can be achieved using long read genome sequencing without parental data, reliable chromosome-scale phasing remains a great challenge. Given that sperm is a natural haploid cell, single-sperm genome sequencing can provide a chromosome-wide phase signal. Due to the limitation of read length, current short-read-based single-sperm genome sequencing methods can only achieve SNP haplotyping and come with difficulties in detecting and haplotyping structural variations (SVs) in complex genomic regions. To overcome these limitations, we developed a long-read-based single-sperm genome sequencing method and a corresponding data analysis pipeline that can accurately identify crossover events and chromosomal level aneuploidies in single sperm and efficiently detect SVs within individual sperm cells. Importantly, without parental genome information, our method can accurately conduct de novo phasing of heterozygous SVs as well as SNPs from male individuals at the whole chromosome scale. The accuracy for phasing of SVs was as high as 98.59% using 100 single sperm cells, and the accuracy for phasing of SNPs was as high as 99.95%. Additionally, our method reliably enabled deduction of the repeat expansions of haplotype-resolved STRs/VNTRs in single sperm cells. Our method provides a new opportunity for studying haplotype-related genetics in mammals.
Sox21 is thought to function as a counteracting partner of SoxB1 ( Sox1, 2, 3 ) genes and is involved in cell fate determination. In this study, we comparatively analyzed the expression patterns and conserved cis‐regulatory elements of the duplicated sox21 genes in zebrafish. In embryogenesis, sox21b is predominantly expressed in the telencephalon, hypothalamus, mesencephalon and lens, and sox21a is solely expressed in the midbrain‐hindbrain boundary, olfactory placode and lateral line, while both genes are expressed in the hindbrain, spinal cord and ear. In adult, sox21a is expressed in the brain, skin, ovary and intestine, while sox21b is expressed in the brain and testis. Interestingly, all 16 pan‐vertebrate conserved non‐coding elements (CNEs) are asymmetrically preserved in the sox21b locus, whereas two fish‐specific elements are kept in the sox21a locus, and this is correlated with increased evolutionary rate of the sox21a protein sequence. Transient transgenic reporter analysis revealed that six sox21b CNEs and two sox21a CNEs drove green fluorescent protein (GFP) expression in tissues correlated with the partitioning of expression in two orthologues. These results indicate that sox21a and sox21b have reciprocally lost expression domains of the ancestral gene reflected by degeneration of certain CNEs in their genomic loci and provide clear evidence for evolution of the duplicated sox21 genes by subfunctionalization. In addition, our data suggest that some CNEs‐based regulatory pathways have been predominantly preserved in the sox21b locus.
Genome assembly has been benefited from long-read sequencing technologies with higher accuracy and higher continuity. However, most human genome assembly require large amount of DNAs from homogeneous cell lines without keeping cell heterogeneities, since cell heterogeneity could profoundly affect haplotype assembly results. Herein, using single-cell genome long-read sequencing technology (SMOOTH-seq), we have sequenced K562 and HG002 cells on PacBio HiFi and Oxford Nanopore Technologies (ONT) platforms and conducted de novo genome assembly. For the first time, we have completed the human genome assembly with high continuity (with NG50 of ∼2 Mb using 95 individual K562 cells) at single-cell levels, and explored the impact of different assemblers and sequencing strategies on genome assembly. With sequencing data from 30 diploid individual HG002 cells of relatively high genome coverage (average coverage ∼41.7%) on ONT platform, the NG50 can reach over 1.3 Mb. Furthermore, with the assembled genome from K562 single-cell dataset, more complete and accurate set of insertion events and complex structural variations could be identified. This study opened a new chapter on the practice of single-cell genome de novo assembly.
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