Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing.

Genome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq to Saccharomyces cerevisiae nuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules.

Peripheral blood TCR clonotype diversity as an age-associated marker of breast cancer progression.

Immune escape is a prerequisite for tumor growth. We previously described a decline in intratumor activated cytotoxic T cells and T cell receptor (TCR) clonotype diversity in invasive breast carcinomas compared to ductal carcinoma in situ (DCIS), implying a central role of decreasing T cell responses in tumor progression. To determine potential associations between peripheral immunity and breast tumor progression, here, we assessed the peripheral blood TCR clonotype of 485 breast cancer patients diagnosed with either DCIS or de novo stage IV disease at younger (<45) or older (≥45) age. TCR clonotype diversity was significantly lower in older compared to younger breast cancer patients regardless of tumor stage at diagnosis. In the younger age group, TCR-α clonotype diversity was lower in patients diagnosed with de novo stage IV breast cancer compared to those diagnosed with DCIS. In the older age group, DCIS patients with higher TCR-α clonotype diversity were more likely to have a recurrence compared to those with lower diversity. Whole blood transcriptome profiles were distinct depending on the TCR-α Chao1 diversity score. There were more CD8+ T cells and a more active immune environment in DCIS tumors of young patients with higher peripheral blood TCR-α Chao1 diversity than in those with lower diversity. These results provide insights into the role that host immunity plays in breast cancer development across different age groups.

Tutorial: integrative computational analysis of bulk RNA-sequencing data to characterize tumor immunity using RIMA.

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification. We describe the RNA-seq IMmune Analysis (RIMA) pipeline integrating many of these tools to streamline RNA-seq analysis. We also developed a comprehensive and user-friendly guide in the form of a GitBook with text and video demos to assist users in analyzing bulk RNA-seq data for immune characterization at both individual sample and cohort levels by using RIMA.

Analysis of visual quality after multifocal intraocular lens implantation in post-LASIK cataract patients.

With the development of refractive corneal surgery, excimer laser in situ keratomileusis (LASIK) has become a common refractive surgery procedure. However, post-LASIK patients are at increased risk of developing cataracts as they age and often require IOL implantation. The choice of IOLs is particularly important for these patients, who have smaller residual refractive error and have higher requirements for post-cataract vision recovery and visual quality than the general population. Multifocal IOLs are widely used in clinical practice for patients with high visual acuity needs, such as cataract patients after refractive keratomileusis, due to their advantages of providing excellent near and distance visual acuity; however, compared to monofocal IOLs, multifocal IOLs can lead to postoperative problems related to visual quality such as increased higher order aberrations and decreased contrast sensitivity. Therefore, whether multifocal IOLs have advantages for post-LASIK cataract patients, such as improving the visual quality of such patients, has attracted attention. In this paper, we analyze the current status of research on the implantation of multifocal IOLs in post-LASIK cataract patients by domestic and foreign experts, review and summarize the relevant literature, and propose further discussion in the context of the actual situation of postoperative visual quality and vision recovery.

Efficient and accurate KIR and HLA genotyping with massively parallel sequencing data.

Killer cell immunoglobulin like receptor (KIR) genes and human leukocyte antigen (HLA) genes play important roles in innate and adaptive immunity. They are highly polymorphic and cannot be genotyped with standard variant calling pipelines. Compared with HLA genes, many KIR genes are similar to each other in sequences and may be absent in the chromosomes. Therefore, although many tools have been developed to genotype HLA genes using common sequencing data, none of them work for KIR genes. Even specialized KIR genotypers could not resolve all the KIR genes. Here we describe T1K, a novel computational method for the efficient and accurate inference of KIR or HLA alleles from RNA-seq, whole-genome sequencing, or whole-exome sequencing data. T1K jointly considers alleles across all genotyped genes, so it can reliably identify present genes and distinguish homologous genes, including the challenging KIR2DL5A/KIR2DL5B genes. This model also benefits HLA genotyping, where T1K achieves high accuracy in benchmarks. Moreover, T1K can call novel single-nucleotide variants and process single-cell data. Applying T1K to tumor single-cell RNA-seq data, we found that KIR2DL4 expression was enriched in tumor-specific CD8+ T cells. T1K may open the opportunity for HLA and KIR genotyping across various sequencing applications.

Role of EPO and TCF7L2 Gene Polymorphism Contribution to the Occurrence of Diabetic Retinopathy.

Objective: For studying the association of EPO (rs551238), EPO (rs1617640), and TCF7L2 (rs7903146) gene with diabetic retinopathy in Northern Chinese population. Methods: We conducted a case-control study, which enrolled 680 subjects and performed SNP genotyping and calculated allele frequencies. Results: When comparison was performed between DR patients and normal persons, the EPO (rs551238) AA genotype has a significant risk association with DR, and AC genotype has a significant protective association with DR. The EPO (rs551238) A allele has a significant risk association with DR, and C allele has a significant protective association with DR. When comparison was performed between DR patients and DM patients, the EPO (rs551238) CC genotype has a significant protective association with DR; the EPO (rs551238) A allele has a significant risk association with DR; and C allele has a significant protective association with DR. When comparison was performed between DR patients and normal persons, the EPO (rs1617640) GT genotype has a significant protective association with DR, and TT genotype has a significant risk association with DR. The EPO (rs1617640) G allele has a significant protective association with DR, and T allele has a significant risk association with DR. In addition, we found that TT genotype does not exist in rs7903146 of TCF7L2 in Chinese population so that the data could not be used. Conclusions: EPO (rs551238, rs1617640) genotype is a susceptible gene for DR in Chinese type 2 diabetic patients, especially the high-risk PDR.

Comprehensive Characterizations of Immune Receptor Repertoire in Tumors and Cancer Immunotherapy Studies.

We applied our computational algorithm TRUST4 to assemble immune receptor (T-cell receptor/B-cell receptor) repertoires from approximately 12,000 RNA sequencing samples from The Cancer Genome Atlas and seven immunotherapy studies. From over 35 million assembled complete complementary-determining region 3 sequences, we observed that the expression of CCL5 and MZB1 is the most positively correlated genes with T-cell clonal expansion and B-cell clonal expansion, respectively. We analyzed amino acid evolution during B-cell receptor somatic hypermutation and identified tyrosine as the preferred residue. We found that IgG1+IgG3 antibodies together with FcRn were associated with complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity or phagocytosis. In addition to B-cell infiltration, we discovered that B-cell clonal expansion and IgG1+IgG3 antibodies are also correlated with better patient outcomes. Finally, we created a website, VisualizIRR, for users to interactively explore and visualize the immune repertoires in this study. See related Spotlight by Liu and Han, p. 786.