Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells.

Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.

Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer.

In this proof-of-concept study, we developed a single-cell method that provides genotypes of somatic alterations found in coding regions of messenger RNAs and integrates these transcript-based variants with their matching cell transcriptomes. We used nanopore adaptive sampling on single-cell complementary DNA libraries to validate coding variants in target gene transcripts, and short-read sequencing to characterize cell types harboring the mutations. CRISPR edits for 16 targets were identified using a cancer cell line, and known variants in the cell line were validated using a 352-gene panel. Variants in primary cancer samples were validated using target gene panels ranging from 161 to 529 genes. A gene rearrangement was also identified in one patient, with the rearrangement occurring in two distinct tumor sites.

Single-molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing.

Epigenetic characterization of cell-free DNA (cfDNA) is an emerging approach for detecting and characterizing diseases such as cancer. We developed a strategy using nanopore-based single-molecule sequencing to measure cfDNA methylomes. This approach generated up to 200 million reads for a single cfDNA sample from cancer patients, an order of magnitude improvement over existing nanopore sequencing methods. We developed a single-molecule classifier to determine whether individual reads originated from a tumor or immune cells. Leveraging methylomes of matched tumors and immune cells, we characterized cfDNA methylomes of cancer patients for longitudinal monitoring during treatment.

Magnetic DNA random access memory with nanopore readouts and exponentially-scaled combinatorial addressing.

The storage of data in DNA typically involves encoding and synthesizing data into short oligonucleotides, followed by reading with a sequencing instrument. Major challenges include the molecular consumption of synthesized DNA, basecalling errors, and limitations with scaling up read operations for individual data elements. Addressing these challenges, we describe a DNA storage system called MDRAM (Magnetic DNA-based Random Access Memory) that enables repetitive and efficient readouts of targeted files with nanopore-based sequencing. By conjugating synthesized DNA to magnetic agarose beads, we enabled repeated data readouts while preserving the original DNA analyte and maintaining data readout quality. MDRAM utilizes an efficient convolutional coding scheme that leverages soft information in raw nanopore sequencing signals to achieve information reading costs comparable to Illumina sequencing despite higher error rates. Finally, we demonstrate a proof-of-concept DNA-based proto-filesystem that enables an exponentially-scalable data address space using only small numbers of targeting primers for assembly and readout.

Wild-type MECP2 expression coincides with age-dependent sensory phenotypes in a female mouse model for Rett syndrome.

Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. Loss of MECP2 protein is thought to cause Rett syndrome phenotypes. The specific underlying mechanisms from typical developmental trajectory to regression features throughout life are unclear. Lack of established timelines to study the molecular, cellular, and behavioral features of regression in female mouse models is a major contributing factor. Due to random X-chromosome inactivation, female patients with Rett syndrome and female mouse models for Rett syndrome (Mecp2Heterozygous , Het) express a functional copy of wild-type MECP2 protein in approximately half of all cells. As MECP2 expression is regulated during early postnatal development and experience, we characterized the expression of wild-type MECP2 in the primary somatosensory cortex of female Het mice. Here, we report increased MECP2 levels in non-parvalbumin-positive neurons of 6-week-old adolescent Het relative to age-matched wild-type controls, while also displaying typical levels of perineuronal net expression in the barrel field subregion of the primary somatosensory cortex, mild tactile sensory perception deficits, and efficient pup retrieval behavior. In contrast, 12-week-old adult Het express MECP2 at levels similar to age-matched wild-type mice, show increased perineuronal net expression in the cortex, and display significant tactile sensory perception deficits. Thus, we have identified a set of behavioral metrics and the cellular substrates to study regression during a specific time in the female Het mouse model, which coincides with changes in wild-type MECP2 expression. We speculate that the precocious increase in MECP2 expression within specific cell types of adolescent Het may provide compensatory benefits at the behavioral level, while the inability to further increase MECP2 levels leads to regressive behavioral phenotypes over time.

Protective Effect of Modified Hemoglobin on Rabbits and Goats in High-Altitude Sickness.

The prevalence and severity of high-altitude sickness increases with increasing altitude. Prevention of hypoxia caused by high-altitude sickness is an urgent problem. As a novel oxygen-carrying fluid, modified hemoglobin can carry oxygen in a full oxygen partial pressure environment and release oxygen in a low oxygen partial pressure environment. It is unclear whether modified hemoglobin can improve hypoxic injury on a plateau. Using hypobaric chamber rabbit (5000 m) and plateau goat (3600 m) models, general behavioral scores and vital signs, hemodynamic, vital organ functions, and blood gas are measured. The results show that the general behavioral scores and vital signs decrease significantly in the hypobaric chamber or plateau, and the modified hemoglobin can effectively improve the general behavioral scores and vital signs in rabbits and goats, and reduce the degree of damage to vital organs. Further studies reveal that arterial partial pressure of oxygen (PaO2 ) and arterial oxygen saturation (SaO2 ) on the plateau decrease rapidly, and the modified hemoglobin could increase PaO2 and SaO2 ; thus, increasing the oxygen-carrying capacity. Moreover, modified hemoglobin has few side effects on hemodynamics and kidney injury. These results indicate that modified hemoglobin has a protective effect against high-altitude sickness.

Single cell and spatial alternative splicing analysis with long read sequencing.

Long-read sequencing has become a powerful tool for alternative splicing analysis. However, technical and computational challenges have limited our ability to explore alternative splicing at single cell and spatial resolution. The higher sequencing error of long reads, especially high indel rates, have limited the accuracy of cell barcode and unique molecular identifier (UMI) recovery. Read truncation and mapping errors, the latter exacerbated by the higher sequencing error rates, can cause the false detection of spurious new isoforms. Downstream, there is yet no rigorous statistical framework to quantify splicing variation within and between cells/spots. In light of these challenges, we developed Longcell, a statistical framework and computational pipeline for accurate isoform quantification for single cell and spatial spot barcoded long read sequencing data. Longcell performs computationally efficient cell/spot barcode extraction, UMI recovery, and UMI-based truncation- and mapping-error correction. Through a statistical model that accounts for varying read coverage across cells/spots, Longcell rigorously quantifies the level of inter-cell/spot versus intra-cell/ spot diversity in exon-usage and detects changes in splicing distributions between cell populations. Applying Longcell to single cell long-read data from multiple contexts, we found that intra-cell splicing heterogeneity, where multiple isoforms co-exist within the same cell, is ubiquitous for highly expressed genes. On matched single cell and Visium long read sequencing for a tissue of colorectal cancer metastasis to the liver, Longcell found concordant signals between the two data modalities. Finally, on a perturbation experiment for 9 splicing factors, Longcell identified regulatory targets that are validated by targeted sequencing.

Colorectal Cancer Metastases in the Liver Establish Immunosuppressive Spatial Networking between Tumor-Associated SPP1+ Macrophages and Fibroblasts.

PURPOSE: The liver is the most frequent metastatic site for colorectal cancer. Its microenvironment is modified to provide a niche that is conducive for colorectal cancer cell growth. This study focused on characterizing the cellular changes in the metastatic colorectal cancer (mCRC) liver tumor microenvironment (TME). EXPERIMENTAL DESIGN: We analyzed a series of microsatellite stable (MSS) mCRCs to the liver, paired normal liver tissue, and peripheral blood mononuclear cells using single-cell RNA sequencing (scRNA-seq). We validated our findings using multiplexed spatial imaging and bulk gene expression with cell deconvolution. RESULTS: We identified TME-specific SPP1-expressing macrophages with altered metabolism features, foam cell characteristics, and increased activity in extracellular matrix (ECM) organization. SPP1+ macrophages and fibroblasts expressed complementary ligand-receptor pairs with the potential to mutually influence their gene-expression programs. TME lacked dysfunctional CD8 T cells and contained regulatory T cells, indicative of immunosuppression. Spatial imaging validated these cell states in the TME. Moreover, TME macrophages and fibroblasts had close spatial proximity, which is a requirement for intercellular communication and networking. In an independent cohort of mCRCs in the liver, we confirmed the presence of SPP1+ macrophages and fibroblasts using gene-expression data. An increased proportion of TME fibroblasts was associated with the worst prognosis in these patients. CONCLUSIONS: We demonstrated that mCRC in the liver is characterized by transcriptional alterations of macrophages in the TME. Intercellular networking between macrophages and fibroblasts supports colorectal cancer growth in the immunosuppressed metastatic niche in the liver. These features can be used to target immune-checkpoint-resistant MSS tumors.

Tumor-associated microbiome features of metastatic colorectal cancer and clinical implications.

Background: Colon microbiome composition contributes to the pathogenesis of colorectal cancer (CRC) and prognosis. We analyzed 16S rRNA sequencing data from tumor samples of patients with metastatic CRC and determined the clinical implications. Materials and methods: We enrolled 133 patients with metastatic CRC at St. Vincent Hospital in Korea. The V3-V4 regions of the 16S rRNA gene from the tumor DNA were amplified, sequenced on an Illumina MiSeq, and analyzed using the DADA2 package. Results: After excluding samples that retained <5% of the total reads after merging, 120 samples were analyzed. The median age of patients was 63 years (range, 34-82 years), and 76 patients (63.3%) were male. The primary cancer sites were the right colon (27.5%), left colon (30.8%), and rectum (41.7%). All subjects received 5-fluouracil-based systemic chemotherapy. After removing genera with <1% of the total reads in each patient, 523 genera were identified. Rectal origin, high CEA level (≥10 ng/mL), and presence of lung metastasis showed higher richness. Survival analysis revealed that the presence of Prevotella (p = 0.052), Fusobacterium (p = 0.002), Selenomonas (p<0.001), Fretibacterium (p = 0.001), Porphyromonas (p = 0.007), Peptostreptococcus (p = 0.002), and Leptotrichia (p = 0.003) were associated with short overall survival (OS, <24 months), while the presence of Sphingomonas was associated with long OS (p = 0.070). From the multivariate analysis, the presence of Selenomonas (hazard ratio [HR], 6.35; 95% confidence interval [CI], 2.38-16.97; p<0.001) was associated with poor prognosis along with high CEA level. Conclusion: Tumor microbiome features may be useful prognostic biomarkers for metastatic CRC.

Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62.

Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Large Cancer Pedigree Involving Multiple Cancer Genes including Likely Digenic MSH2 and MSH6 Lynch Syndrome (LS) and an Instance of Recombinational Rescue from LS.

Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.

Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing.

We developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.

The Protective Effect of a Novel Cross-Linked Hemoglobin-Based Oxygen Carrier on Hypoxia Injury of Acute Mountain Sickness in Rabbits and Goats.

Hypoxia is the major cause of acute altitude hypoxia injury in acute mountain sickness (AMS). YQ23 is a kind of novel bovine-derived, cross-linked hemoglobin-based oxygen carrier (HBOC). It has an excellent capacity for carrying and releasing oxygen. Whether YQ23 has a protective effect on the acute altitude hypoxia injury in AMS is unclear. In investigating this mechanism, the hypobaric chamber rabbit model and plain-to-plateau goat model were used. Furthermore, this study measured the effects of YQ23 on the ability of general behavior, general vital signs, Electrocardiograph (ECG), hemodynamics, vital organ injury markers, and blood gases in hypobaric chamber rabbits and plain-to-plateau goats. Our results showed that the ability of general behavior (general behavioral scores, GBS) (GBS: 18 ± 0.0 vs. 14 ± 0.5, p < 0.01) and the general vital signs weakened [Heart rate (HR, beats/min): 253.5 ± 8.7 vs. 301.1 ± 19.8, p < 0.01; Respiratory rate (RR, breaths/min): 86.1 ± 5.2 vs. 101.2 ± 7.2, p < 0.01] after exposure to plateau environment. YQ23 treatment significantly improved the ability of general behavior (GBS: 15.8 ± 0.5 vs. 14.0 ± 0.5, p < 0.01) and general vital signs [HR (beats/min): 237.8 ± 24.6 vs. 301.1 ± 19.8, p < 0.01; RR (breaths/min): 86.9 ± 6.6 vs. 101.2 ± 7.2, p < 0.01]. The level of blood PaO2 (mmHg) (115.3 ± 4.7 vs. 64.2 ± 5.6, p < 0.01) and SaO2(%) (97.7 ± 0.7 vs. 65.8 ± 3.1, p < 0.01) sharply decreased after exposure to plateau, YQ23 treatment significantly improved the blood PaO2 (mmHg) (97.6 ± 3.7 vs. 64.2 ± 5.6, p < 0.01) and SaO2(%) (82.7 ± 5.2 vs. 65.8 ± 3.1, p < 0.01). The cardiac ischemia and injury marker was increased [troponin (TnT, µg/L):0.08 ± 0.01 vs. 0.12 ± 0.02, p < 0.01], as well as the renal [blood urea nitrogen (BUN, mmol/L): 6.0 ± 0.7 vs. 7.3 ± 0.5, p < 0.01] and liver injury marker [alanine aminotransferase (ALT, U/L): 45.8 ± 3.6 vs. 54.6 ± 4.2, p < 0.01] was increased after exposure to a plateau environment. YQ23 treatment markedly alleviated cardiac ischemia [TnT (µg/L):0.10 ± 0.01 vs 0.12 ± 0.02, p < 0.01] and mitigated the vital organ injury. Besides, YQ23 exhibited no adverse effects on hemodynamics, myocardial ischemia, and renal injury. In conclusion, YQ23 effectively alleviates acute altitude hypoxia injury of AMS without aside effects.

Oxytocin receptors in the midbrain dorsal raphe are essential for postpartum maternal social and affective behaviors.

Oxytocin receptors (OTRs) in the midbrain dorsal raphe (DR; the source of most forebrain serotonin) have recently been identified as a potential pharmacological target for treating numerous psychiatric disorders. However, almost all research on this topic has been conducted on males and the role of DR OTRs in female social and affective behaviors is mostly unknown. This may be particularly relevant during early motherhood, which is a time of high endogenous oxytocin signaling, but also a time of elevated risk for psychiatric dysfunction. To investigate whether OTRs in the DR are necessary for postpartum female social and affective behaviors, we constructed and then injected into the DR an adeno-associated virus permanently expressing an shRNA targeting OTR mRNA. We then observed a suite of social and affective behaviors postpartum. OTR knockdown in the maternal DR led to pup loss after parturition, decreased nursing, increased aggression, and increased behavioral despair. These effects of OTR knockdown in the DR may be due to disrupted neuroplasticity in the primary somatosensory cortex (S1), which mediates maternal sensitivity to the tactile cues from young, as we found significantly more plasticity-restricting perineuronal nets (PNNs) in the S1 rostral barrel field and fewer PNNs in the caudal barrel field of OTR-knockdown mothers. These results demonstrate that OTRs in the midbrain DR are essential for postpartum maternal social and affective behaviors, are involved in postpartum cortical plasticity, and suggest that pharmacotherapies targeting OTRs in the DR could be effective treatments for some peripartum affective disorders.

A Novel Cross-Linked Hemoglobin-Based Oxygen Carrier, YQ23, Extended the Golden Hour for Uncontrolled Hemorrhagic Shock in Rats and Miniature Pigs.

Background: Hypotensive resuscitation is widely applied for trauma and war injury to reduce bleeding during damage-control resuscitation, but the treatment time window is limited in order to avoid hypoxia-associated organ injury. Whether a novel hemoglobin-based oxygen carrier (HBOC), YQ23 in this study, could protect organ function, and extend the Golden Hour for treatment is unclear. Method: Uncontrolled hemorrhagic shock rats and miniature pigs were infused with 0.5, 2, and 5% YQ23 before bleeding was controlled, while Lactate Ringer's solution (LR) and fresh whole blood plus LR (WB + LR) were set as controls. During hypotensive resuscitation the mean blood pressure was maintained at 50-60 mmHg for 60 min. Hemodynamics, oxygen delivery and utilization, blood loss, fluid demand, organ function, animal survival as well as side effects were observed. Besides, in order to observe whether YQ23 could extend the Golden Hour, the hypotensive resuscitation duration was extended to 180 min and animal survival was observed. Results: Compared with LR, infusion of YQ23 in the 60 min pre-hospital hypotensive resuscitation significantly reduced blood loss and the fluid demand in both rats and pigs. Besides, YQ23 could effectively stabilize hemodynamics, and increase tissue oxygen consumption, increase the cardiac output, reduce liver and kidney injury, which helped to reduce the early death and improve animal survival. In addition, the hypotensive resuscitation duration could be extended to 180 min using YQ23. Side effects such as vasoconstriction and renal injury were not observed. The beneficial effects of 5% YQ23 are equivalent to similar volume of WB + LR. Conclusion: HBOC, such as YQ23, played vital roles in damage-control resuscitation for emergency care and benefited the uncontrolled hemorrhagic shock in the pre-hospital treatment by increasing oxygen delivery, reducing organ injury. Besides, HBOC could benefit the injured and trauma patients by extending the Golden Hour.

Systematic analysis of goal-related movement sequences during maternal behaviour in a female mouse model for Rett syndrome.

Rodent dams seek and gather scattered pups back to the nest (pup retrieval), an essential aspect of maternal care. Systematic analysis of the dynamic sequences of goal-related movements that comprise the entire behavioural sequence, which would be ultimately essential for understanding the underlying neurobiology, is not well-characterized. Here, we present such analysis across 3 days in alloparental female mice (Surrogates or Sur) of two genotypes; Mecp2Heterozygotes (Het), a female mouse model for Rett syndrome and their wild type (WT) siblings. We analysed CBA/CaJ and C57BL/6J WT surrogates for within-strain comparisons. Frame-by-frame analysis over different phases was performed manually using DataVyu software. We previously showed that surrogate Het are inefficient at pup retrieval, by end-point analysis such as latency index and errors. Here, the sequence of searching, pup-approach and successful retrieval streamlines over days for WT, while Het exhibits variations in this pattern. Goal-related movements between Het and WT are similar in other phases, suggesting context-driven atypical patterns in Het during the pup retrieval phase. We identified proximal pup approach and pup grooming as atypical tactile interactions between pups and Het. Day-by-day analysis showed dynamic changes in goal-related movements in individual animals across genotypes and strains. Overall, our approach (1) highlights natural variation in individual mice on different days, (2) establishes a "gold-standard" manually curated dataset to help build behavioural repertoires using machine learning approaches, and (3) suggests atypical tactile sensory processing and possible regression in a female mouse model for Rett syndrome.

Integrative single-cell analysis of allele-specific copy number alterations and chromatin accessibility in cancer.

Cancer progression is driven by both somatic copy number aberrations (CNAs) and chromatin remodeling, yet little is known about the interplay between these two classes of events in shaping the clonal diversity of cancers. We present Alleloscope, a method for allele-specific copy number estimation that can be applied to single-cell DNA- and/or transposase-accessible chromatin-sequencing (scDNA-seq, ATAC-seq) data, enabling combined analysis of allele-specific copy number and chromatin accessibility. On scDNA-seq data from gastric, colorectal and breast cancer samples, with validation using matched linked-read sequencing, Alleloscope finds pervasive occurrence of highly complex, multiallelic CNAs, in which cells that carry varying allelic configurations adding to the same total copy number coevolve within a tumor. On scATAC-seq from two basal cell carcinoma samples and a gastric cancer cell line, Alleloscope detected multiallelic copy number events and copy-neutral loss-of-heterozygosity, enabling dissection of the contributions of chromosomal instability and chromatin remodeling to tumor evolution.

Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies.

BACKGROUND: The genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contact tracing. METHODS: Leveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies variants, and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints. RESULTS: We built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and a combination of mutations that appear in only a small number of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of 100 SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome. CONCLUSIONS: We conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients and determined their general prevalence when compared to over 70,000 other strains. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies.

Background: The genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contract tracing. Methods: Leveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints. Results: We built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and rare ones that occur in only small fraction of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome. Conclusions: We conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients, mutations demarcating dominant species and the prevalence of mutation signatures, of which a significant number were relatively unique. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2.

Cortical neuronal circuits along the sensorimotor pathways are shaped by experience during critical periods of heightened plasticity in early postnatal development. After closure of critical periods, measured histologically by the formation and maintenance of extracellular matrix structures called perineuronal nets (PNNs), the adult mouse brain exhibits restricted plasticity and maturity. Mature PNNs are typically considered to be stable structures that restrict synaptic plasticity on cortical parvalbumin+ (PV+) GABAergic neurons. Changes in environment (i.e., novel behavioral training) or social contexts (i.e., motherhood) are known to elicit synaptic plasticity in relevant neural circuitry. However, little is known about concomitant changes in the PNNs surrounding the cortical PV+ GABAergic neurons. Here, we show novel changes in PNN density in the primary somatosensory cortex (SS1) of adult female mice after maternal experience [called surrogate (Sur)], using systematic microscopy analysis of a whole brain region. On average, PNNs were increased in the right barrel field and decreased in the left forelimb regions. Individual mice had left hemisphere dominance in PNN density. Using adult female mice deficient in methyl-CpG-binding protein 2 (MECP2), an epigenetic regulator involved in regulating experience-dependent plasticity, we found that MECP2 is critical for this precise and dynamic expression of PNN. Adult naive Mecp2-heterozygous (Het) females had increased PNN density in specific subregions in both hemispheres before maternal experience, compared with wild-type (WT) littermate controls. The laterality in PNN expression seen in naive Het (NH) was lost after maternal experience in Sur Het (SH) mice, suggesting possible intact mechanisms for plasticity. Together, our results identify subregion and hemisphere-specific alterations in PNN expression in adult females, suggesting extracellular matrix plasticity as a possible neurobiological mechanism for adult behaviors in rodents.