sciMET-cap: high-throughput single-cell methylation analysis with a reduced sequencing burden.

DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Accumulated off-target coverage enables genome-wide differentially methylated region (DMR) calling for clusters with as few as 115 cells. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Factors that influence performance in pediatric cochlear implant recipients with cochlear nerve deficiency.

OBJECTIVES: Children with cochlear nerve deficiency (CND) have wide variability in outcomes with cochlear implant (CI) use. The current study aims to report a large cohort of pediatric CI recipients with CND and to evaluate for factors that may predict improved performance. METHODS: The current study is a retrospective review of pediatric CI recipients with CND at a tertiary academic hospital. Variables including cochlear nerve status (hypoplasia vs aplasia), age at implantation, cochleovestibular malformation, bony cochlear nerve aperture, internal auditory canal aperture, and cognitive delay were evaluated for predictors of postoperative performance. A stepwise multinomial regression analysis was performed. RESULTS: Forty-seven CI recipients (54 ears) were included in the analysis. A majority (59%) showed auditory capabilities with their CI. Twenty percent of recipients achieved some level of open-set speech perception with their CI. The regression analysis identified cochlear nerve status and cognitive delay as predictors of performance. CI recipients with cochlear nerve hypoplasia had significantly improved performance compared to those with aplasia (p = 0.003). Recipients with cognitive delay had more limited benefit than those without cognitive delay (p = 0.033). CONCLUSIONS: Children with CND can benefit from CI use, with outcomes spanning from non-use to development of spoken language. Predictive factors for improved performance include a lack of cognitive delay and cochlear hypoplasia rather than aplasia. These can be important considerations for parent counseling and decision making.

Accessible high-throughput single-cell whole-genome sequencing with paired chromatin accessibility.

Single-cell whole-genome sequencing (scWGS) enables the assessment of genome-level molecular differences between individual cells with particular relevance to genetically diverse systems like solid tumors. The application of scWGS was limited due to a dearth of accessible platforms capable of producing high-throughput profiles. We present a technique that leverages nucleosome disruption methodologies with the widely adopted 10× Genomics ATAC-seq workflow to produce scWGS profiles for high-throughput copy-number analysis without new equipment or custom reagents. We further demonstrate the use of commercially available indexed transposase complexes from ScaleBio for sample multiplexing, reducing the per-sample preparation costs. Finally, we demonstrate that sequential indexed tagmentation with an intervening nucleosome disruption step allows for the generation of both ATAC and WGS data from the same cell, producing comparable data to the unimodal assays. By exclusively utilizing accessible commercial reagents, we anticipate that these scWGS and scWGS+ATAC methods can be broadly adopted by the research community.

sciMET-cap: High-throughput single-cell methylation analysis with a reduced sequencing burden.

DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Sufficient off-target coverage further enables the production of near-complete methylomes for individual cell types. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

Comparison of Two Place-Based Mapping Procedures on Masked Sentence Recognition as a Function of Electrode Array Angular Insertion Depth and Presence of Acoustic Low-Frequency Information: A Simulation Study.

INTRODUCTION: Cochlear implant (CI) and electric-acoustic stimulation (EAS) users may experience better performance with maps that align the electric filter frequencies to the cochlear place frequencies, known as place-based maps, than with maps that present spectrally shifted information. Individual place-based mapping procedures differ in the frequency content that is aligned to cochlear tonotopicity versus discarded or spectrally shifted. The performance benefit with different place-based maps may vary due to individual differences in angular insertion depth (AID) of the electrode array and whether functional acoustic low-frequency information is available in the implanted ear. The present study compared masked speech recognition with two types of place-based maps as a function of AID and presence of acoustic low-frequency information. METHODS: Sixty adults with normal hearing listened acutely to CI or EAS simulations of two types of place-based maps for one of three cases of electrode arrays at shallow AIDs. The strict place-based (Strict-PB) map aligned the low- and mid-frequency information to cochlear tonotopicity and discarded information below the frequency associated with the most apical electrode contact. The alternative place-based map (LFshift-PB) aligned the mid-frequency information to cochlear tonotopicity and provided more of the speech spectrum by compressing low-frequency information on the apical electrode contacts (i.e., <1 kHz). Three actual cases of a 12-channel, 24-mm electrode array were simulated by assigning the carrier frequency for an individual channel as the cochlear place frequency of the associated electrode contact. The AID and cochlear place frequency for the most apical electrode contact were 460° and 498 Hz for case 1, 389° and 728 Hz for case 2, and 335° and 987 Hz for case 3, respectively. RESULTS: Generally, better performance was observed with the Strict-PB maps for cases 1 and 2, where mismatches were 2-4 octaves for the most apical channel with the LFshift-PB map. Similar performance was observed between maps for case 3. For the CI simulations, performance with the Strict-PB map declined with decreases in AID, while performance with the LFshift-PB map remained stable across cases. For the EAS simulations, performance with the Strict-PB map remained stable across cases, while performance with the LFshift-PB map improved with decreases in AID. CONCLUSIONS: Listeners demonstrated differences with the Strict-PB versus LFshift-PB maps as a function of AID and whether acoustic low-frequency information was available (CI vs. EAS). These data support the use of the Strict-PB mapping procedure for AIDs ≥335°, though further study including time for acclimatization in CI and EAS users is warranted.

Atlas-scale single-cell chromatin accessibility using nanowell-based combinatorial indexing.

Here we present advancements in single-cell combinatorial indexed Assay for Transposase Accessible Chromatin (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>105 cells) at low cost. The platform leverages the core of the sciATAC workflow where multiple indexed tagmentation reactions are performed, followed by pooling and distribution to a second set of reaction wells for polymerase chain reaction (PCR)-based indexing. In this work, we instead leverage a chip containing 5184 nanowells at the PCR stage of indexing, enabling a 52-fold improvement in scale and reduction in per-cell preparation costs. We detail three variants that balance cell throughput and depth of coverage, and apply these methods to banked mouse brain tissue, producing maps of cell types as well as neuronal subtypes that include integration with existing single-cell Assay for Transposase Accessible Chromatin (scATAC) and scRNA-seq data sets. Our optimized workflow achieves a high fraction of reads that fall within called peaks (>80%) and low cell doublet rates. The high cell coverage technique produces high unique reads per cell, while retaining high enrichment for open chromatin regions, enabling the assessment of >70,000 unique accessible loci on average for each cell profiled. When compared to current methods in the field, our technique provides similar or superior per-cell information with very low levels of cell-to-cell cross talk, and achieves this at a cost point much lower than existing assays.

Influence of Electric Frequency-to-Place Mismatches on the Early Speech Recognition Outcomes for Electric-Acoustic Stimulation Users.

PURPOSE: Cochlear implant (CI) recipients with hearing preservation experience significant improvements in speech recognition with electric-acoustic stimulation (EAS) as compared to with a CI alone, although outcomes across EAS users vary. The individual differences in performance may be due in part to default mapping procedures, which result in electric frequency-to-place mismatches for the majority of EAS users. This study assessed the influence of electric mismatches on the early speech recognition for EAS users. METHOD: Twenty-one participants were randomized at EAS activation to listen exclusively with a default or place-based map. For both groups, the unaided thresholds determined the acoustic cutoff frequency (i.e., > 65 dB HL). For default maps, the electric filter frequencies were assigned to avoid spectral gaps in frequency information but created varying magnitudes of mismatches. For place-based maps, the electric filter frequencies were assigned to avoid frequency-to-place mismatches. Recognition of consonant-nucleus-consonant words and vowels was assessed at activation and 1, 3, and 6 months postactivation. RESULTS: For participants with default maps, electric mismatch at 1500 Hz ranged from 2 to -12.0 semitones (Mdn = -5 semitones). Poorer performance was observed for those with larger magnitudes of electric mismatch. This effect was observed through 6 months of EAS listening experience. CONCLUSIONS: The present sample of EAS users experienced better initial performance when electric mismatches were small or eliminated. These data suggest the utility of methods that reduce electric mismatches, such as place-based mapping procedures. Investigation is ongoing to determine whether these differences persist with long-term EAS use. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.22096523.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Safety of sequential immune checkpoint inhibitors after prior immune therapy.

BACKGROUND: The use of immune checkpoint inhibitors (ICI) has transformed cancer treatment. Subsequent ICI use has become increasingly common following disease progression. We aim to evaluate the safety and tolerability of the sequential ICI treatment modality. METHODS: Retrospective review of confirmed carcinoma from January 2014 to December 2018. Patients were categorized into "initial ICI arm" and "sequential ICI arm" defined as patients receiving single, dual or chemo-immunotherapy ICI following an initial ICI regimen. Primary outcome was the development of a new or recurrent immune related adverse event (irAE) during sequential therapy. Secondary outcomes were the number of cycles prior to the development of irAE and grade of irAE. RESULTS: A total of 483 patients received ICI during the timeframe. Of those, 22 patients received sequential ICI. The diagnoses included ten lung cancer, seven melanoma, four renal cell carcinoma and one bladder cancer. 16 patients received single agent ICI following the initial ICI, three patients received dual ICI following the initial ICI, one patient received chemotherapy-immunotherapy following initial ICI, and two patients received chemo-immunotherapy after dual ICI. Four patients developed new irAE and one patient developed the same irAE on sequential treatment. A higher proportion of patients experienced grade 3 irAE in the sequential arm compared to the initial ICI arm (p = 0.03). No statistical difference was found between the development of irAE and the number of cycles prior to development of irAE in either treatment groups (p = 0.5). CONCLUSION: Our data shows overall safety of sequencing ICI when close monitoring was employed.

High-throughput robust single-cell DNA methylation profiling with sciMETv2.

DNA methylation is a key epigenetic property that drives gene regulatory programs in development and disease. Current single-cell methods that produce high quality methylomes are expensive and low throughput without the aid of extensive automation. We previously described a proof-of-principle technique that enabled high cell throughput; however, it produced only low-coverage profiles and was a difficult protocol that required custom sequencing primers and recipes and frequently produced libraries with excessive adapter contamination. Here, we describe a greatly improved version that generates high-coverage profiles (~15-fold increase) using a robust protocol that does not require custom sequencing capabilities, includes multiple stopping points, and exhibits minimal adapter contamination. We demonstrate two versions of sciMETv2 on primary human cortex, a high coverage and rapid version, identifying distinct cell types using CH methylation patterns. These datasets are able to be directly integrated with one another as well as with existing snmC-seq2 datasets with little discernible bias. Finally, we demonstrate the ability to determine cell types using CG methylation alone, which is the dominant context for DNA methylation in most cell types other than neurons and the most applicable analysis outside of brain tissue.

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.

Effect of Place-Based Versus Default Mapping Procedures on Masked Speech Recognition: Simulations of Cochlear Implant Alone and Electric-Acoustic Stimulation.

PURPOSE: Cochlear implant (CI) recipients demonstrate variable speech recognition when listening with a CI-alone or electric-acoustic stimulation (EAS) device, which may be due in part to electric frequency-to-place mismatches created by the default mapping procedures. Performance may be improved if the filter frequencies are aligned with the cochlear place frequencies, known as place-based mapping. Performance with default maps versus an experimental place-based map was compared for participants with normal hearing when listening to CI-alone or EAS simulations to observe potential outcomes prior to initiating an investigation with CI recipients. METHOD: A noise vocoder simulated CI-alone and EAS devices, mapped with default or place-based procedures. The simulations were based on an actual 24-mm electrode array recipient, whose insertion angles for each electrode contact were used to estimate the respective cochlear place frequency. The default maps used the filter frequencies assigned by the clinical software. The filter frequencies for the place-based maps aligned with the cochlear place frequencies for individual contacts in the low- to mid-frequency cochlear region. For the EAS simulations, low-frequency acoustic information was filtered to simulate aided low-frequency audibility. Performance was evaluated for the AzBio sentences presented in a 10-talker masker at +5 dB signal-to-noise ratio (SNR), +10 dB SNR, and asymptote. RESULTS: Performance was better with the place-based maps as compared with the default maps for both CI-alone and EAS simulations. For instance, median performance at +10 dB SNR for the CI-alone simulation was 57% correct for the place-based map and 20% for the default map. For the EAS simulation, those values were 59% and 37% correct. Adding acoustic low-frequency information resulted in a similar benefit for both maps. CONCLUSIONS: Reducing frequency-to-place mismatches, such as with the experimental place-based mapping procedure, produces a greater benefit in speech recognition than maximizing bandwidth for CI-alone and EAS simulations. Ongoing work is evaluating the initial and long-term performance benefits in CI-alone and EAS users. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.19529053.

Deeply conserved synteny and the evolution of metazoan chromosomes.

Animal genomes show networks of deeply conserved gene linkages whose phylogenetic scope and chromosomal context remain unclear. Here, we report chromosome-scale conservation of synteny among bilaterians, cnidarians, and sponges and use comparative analysis to reconstruct ancestral chromosomes across major animal groups. Comparisons among diverse metazoans reveal the processes of chromosome evolution that produced contemporary karyotypes from their Precambrian progenitors. On the basis of these findings, we introduce a simple algebraic representation of chromosomal change and use it to establish a unified systematic framework for metazoan chromosome evolution. We find that fusion-with-mixing, a previously unappreciated mode of chromosome change, has played a central role. We find that relicts of several metazoan chromosomal units are preserved in unicellular eukaryotes. These conserved pre-metazoan linkages include the chromosomal unit that encodes the most diverse set of metazoan homeobox genes, suggesting a candidate genomic context for the early diversification of this key gene family.

Relationship Between Intraoperative Electrocochleography and Hearing Preservation.

OBJECTIVES: To compare intraoperative intracochlear electrocochleography (ECochG) with hearing preservation outcomes in cochlear implant (CI) subjects. DESIGN: Intraoperative electrocochleography was performed in adult CI subjects who were recipients of Advanced Bionics' Bionics LLC precurved HiFocus MidScala or straight HiFocus SlimJ electrode arrays. ECochG responses were recorded from the most apical electrode contact during insertion. No changes to the insertions were made due to ECochG monitoring. No information about insertion resistance was collected. ECochG drops were estimated as the change in amplitude from peak (defined as maximum amplitude response) to drop (largest drop) point after the peak during insertion was measured following the peak response. Audiometric thresholds from each subject were obtained before and approximately 1 month after CI surgery. The change in pure tone average for frequencies between 125 Hz and 500 Hz was measured after surgery. No postoperative CT scans were collected as part of this study. RESULTS: A total of 68 subjects from five surgical centers participated in the study. The study sample included 30 MidScala and 38 SlimJ electrodes implanted by approximately 20 surgeons who contributed to the study. Although a wide range of results were observed, there was a moderate positive correlation (Pearson Correlation coefficient, r = 0.56, p < 0.01) between the size of the ECochG drop and the magnitude of pure tone average change. This trend was present for both the MidScala and SlimJ arrays. The SlimJ and MidScala arrays produced significantly different hearing loss after surgery. CONCLUSION: Large ECochG amplitude drops observed during electrode insertion indicated poorer hearing preservation. Although the outcomes were variable, this information may be helpful to guide surgical decision-making when contemplating full electrode insertion and the likelihood of hearing preservation.

Insertion Depth and Cochlear Implant Speech Recognition Outcomes: A Comparative Study of 28- and 31.5-mm Lateral Wall Arrays.

OBJECTIVES: 1) To compare speech recognition outcomes between cochlear implant (CI) recipients of 28- and 31.5-mm lateral wall electrode arrays, and 2) to characterize the relationship between angular insertion depth (AID) and speech recognition. STUDY DESIGN: Retrospective review. SETTING: Tertiary academic referral center. PATIENTS: Seventy-five adult CI recipients of fully inserted 28-mm (n = 28) or 31.5-mm (n = 47) lateral wall arrays listening with a CI-alone device. INTERVENTIONS: Cochlear implantation with postoperative computed tomography. MAIN OUTCOME MEASURES: Consonant-nucleus-consonant (CNC) word recognition assessed with the CI-alone at 12 months postactivation. RESULTS: The mean AID of the most apical electrode contact for the 31.5-mm array recipients was significantly deeper than the 28-mm array recipients (628° vs 571°, p < 0.001). Following 12 months of listening experience, mean CNC word scores were significantly better for recipients of 31.5-mm arrays compared with those implanted with 28-mm arrays (59.5% vs 48.3%, p = 0.004; Cohen's d = 0.70; 95% CI [0.22, 1.18]). There was a significant positive correlation between AID and CNC word scores (r = 0.372, p = 0.001), with a plateau in performance observed around 600°. CONCLUSIONS: Cochlear implant recipients implanted with a 31.5-mm array experienced better speech recognition than those with a 28-mm array at 12 months postactivation. Deeper insertion of a lateral wall array appears to confer speech recognition benefit up to ∼600°, with a plateau in performance observed thereafter. These data provide preliminary evidence of the insertion depth necessary to optimize speech recognition outcomes for lateral wall electrode arrays among CI-alone users.

Influence of Postponed Follow-Up after Cochlear Implant Activation during the COVID-19 Pandemic on Aided Sound Field Detection and Speech Recognition.

INTRODUCTION: The objective of this study was to assess the influence of postponing the first post-activation follow-up due to the COVID-19 pandemic on the aided sound field detection thresholds and speech recognition of cochlear implant (CI) users. METHODS: A retrospective review was performed at a tertiary referral center. Two groups of adult CI recipients were evaluated: (1) patients whose first post-activation follow-up was postponed due to COVID-19 closures (postponed group; n = 10) and (2) a control group that attended recommended post-activation follow-ups prior to the COVID-19 pandemic (control group; n = 18). For both groups, electric thresholds were estimated at initial activation based on comfort levels and were measured behaviorally at subsequent post-activation follow-ups. For the control group, behavioral thresholds were measured at the 1-month follow-up. For the postponed group, behavioral thresholds were not measured until 3 months post-activation since the 1-month follow-up was postponed. The aided pure-tone average (PTA) and word recognition results were compared between groups at the 3-month follow-up and at an interim visit 2-9 weeks later. RESULTS: At the 3-month follow-up, the postponed group had significantly poorer word recognition (23 vs. 42%, p = 0.027) and aided PTA (42 vs. 37 dB HL, p = 0.041) than the control group. No significant differences were observed between 3-month data from the control group and interim data from the postponed group. CONCLUSIONS: The postponed follow-up after CI activation was associated with poorer outcomes, both in terms of speech recognition and aided audibility. However, these detrimental effects were reversed following provision of an individualized map, with behaviorally measured electric threshold and comfort levels. While adult CI recipients demonstrate an improvement in speech recognition with estimated electric thresholds, the present results suggest that behavioral mapping within the initial weeks of device use may support optimal outcomes.

Influence of Cochlear Implant Use on Perceived Listening Effort in Adult and Pediatric Cases of Unilateral and Asymmetric Hearing Loss.

OBJECTIVE: Assess the influence of cochlear implant (CI) use on the perceived listening effort of adult and pediatric subjects with unilateral hearing loss (UHL) or asymmetric hearing loss (AHL). STUDY DESIGN: Prospective cohort. SETTING: Tertiary referral center. PATIENTS: Adults and children with UHL or AHL. INTERVENTION: Cochlear implantation. Subjects received their CI as part of a clinical trial assessing the effectiveness of cochlear implantation in cases of UHL and AHL. MAIN OUTCOME MEASURES: Responses to the Listening Effort pragmatic subscale on the Speech, Spatial, and Qualities of Hearing Scale (SSQ) or SSQ for Children with Impaired Hearing (SSQ-C) were compared over the study period. Subjects or their parents completed the questionnaires preoperatively and at predetermined postactivation intervals. For the adult subjects, responses were compared to word recognition in quiet and sentence recognition in noise. RESULTS: Forty adult subjects (n = 20 UHL, n = 20 AHL) and 16 pediatric subjects with UHL enrolled and underwent cochlear implantation. Subjects in all three groups reported a significant reduction in perceived listening effort within the initial months of CI use (p < 0.001; η2 ≥ 0.351). The perceived benefit was significantly correlated with speech recognition in noise for the adult subjects with UHL at the 12-month interval (r(20) = .59, p = 0.006). CONCLUSIONS: Adult and pediatric CI recipients with UHL or AHL report a reduction in listening effort with CI use as compared to their preoperative experiences. Use of the SSQ and SSQ-C Listening Effort pragmatic subscale may provide additional information about a CI recipient's experience beyond the abilities measured in the sound booth.