TAGET: a toolkit for analyzing full-length transcripts from long-read sequencing.

Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis. We present an integrated computational toolkit named TAGET for Iso-seq full-length transcript data analyses, including transcript alignment, annotation, gene fusion detection, and quantification analyses such as differential expression gene analysis and differential isoform usage analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.

Comprehensive analysis of neoantigens derived from structural variation across whole genomes from 2528 tumors.

BACKGROUND: Neoantigens are critical for anti-tumor immunity and have been long-envisioned as promising therapeutic targets. However, current neoantigen analyses mostly focus on single nucleotide variations (SNVs) and indel mutations and seldom consider structural variations (SVs) that are also prevalent in cancer. RESULTS: Here, we develop a computational method termed NeoSV, which incorporates SV annotation, protein fragmentation, and MHC binding prediction together, to predict SV-derived neoantigens. Analysis of 2528 whole genomes reveals that SVs significantly contribute to the neoantigen repertoire in both quantity and quality. Whereas most neoantigens are patient-specific, shared neoantigens are identified with high occurrence rates in breast, ovarian, and gastrointestinal cancers. We observe extensive immunoediting on SV-derived neoantigens, especially on clonal events, which suggests their immunogenic potential. We also demonstrate that genomic alteration-related neoantigen burden, which integrates SV-derived neoantigens, depicts the tumor-immune interplay better than tumor neoantigen burden and may improve patient selection for immunotherapy. CONCLUSIONS: Our study fills the gap in the current neoantigen repertoire and provides a valuable resource for cancer vaccine development.

An extra-clock ultradian brain oscillator sustains circadian timekeeping.

The master circadian clock generates 24-hour rhythms to orchestrate daily behavior, even running freely under constant conditions. Traditionally, the master clock is considered self-sufficient in sustaining free-running timekeeping via its cell-autonomous molecular clocks and interneuronal communications within the circadian neural network. Here, we find a set of bona fide ultradian oscillators in the Drosophila brain that support free-running timekeeping, despite being located outside the master clock circuit and lacking clock gene expression. These extra-clock electrical oscillators (xCEOs) generate cell-autonomous ultradian bursts, pacing widespread burst firing and promoting rhythmic resting membrane potentials in clock neurons via parallel monosynaptic connections. Silencing xCEOs disrupts daily electrical rhythms in clock neurons and impairs cycling of neuropeptide pigment dispersing factor, leading to the loss of free-running locomotor rhythms. Together, we conclude that the master clock is not self-sufficient to sustain free-running behavior rhythms but requires additional endogenous inputs to the clock from the extra-clock ultradian brain oscillators.

Drosulfakinin signaling modulates female sexual receptivity in Drosophila.

Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) to promote female sexual behavior in Drosophila. Loss of DSK function reduces female receptivity while overexpressing DSK enhances female receptivity. We identify two pairs of Dsk-expressing neurons in the central brain to promote female receptivity. We find that the DSK peptide acts through one of its receptors, CCKLR-17D3, to modulate female receptivity. Manipulation of CCKLR-17D3 and its expressing neurons alters female receptivity. We further reveal that the two pairs of Dsk-expressing neurons receive input signal from pC1 neurons that integrate sex-related cues and mating status. These results demonstrate how a neuropeptide pathway interacts with a central neural node in the female sex circuitry to modulate sexual receptivity.

Odor-evoked inhibition of olfactory sensory neurons drives olfactory perception in Drosophila.

Inhibitory response occurs throughout the nervous system, including the peripheral olfactory system. While odor-evoked excitation in peripheral olfactory cells is known to encode odor information, the molecular mechanism and functional roles of odor-evoked inhibition remain largely unknown. Here, we examined Drosophila olfactory sensory neurons and found that inhibitory odors triggered outward receptor currents by reducing the constitutive activities of odorant receptors, inhibiting the basal spike firing in olfactory sensory neurons. Remarkably, this odor-evoked inhibition of olfactory sensory neurons elicited by itself a full range of olfactory behaviors from attraction to avoidance, as did odor-evoked olfactory sensory neuron excitation. These results indicated that peripheral inhibition is comparable to excitation in encoding sensory signals rather than merely regulating excitation. Furthermore, we demonstrated that a bidirectional code with both odor-evoked inhibition and excitation in single olfactory sensory neurons increases the odor-coding capacity, providing a means of efficient sensory encoding.

Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons.

In Drosophila, olfactory sensory neurons (OSNs) rely primarily on two types of chemoreceptors, odorant receptors (Ors) and ionotropic receptors (Irs), to convert odor stimuli into neural activity. The cellular signaling of these receptors in their native OSNs remains unclear because of the difficulty of obtaining intracellular recordings from Drosophila OSNs. Here, we developed an antennal preparation that enabled the first recordings (to our knowledge) from targeted Drosophila OSNs through a patch-clamp technique. We found that brief odor pulses triggered graded inward receptor currents with distinct response kinetics and current-voltage relationships between Or- and Ir-driven responses. When stimulated with long-step odors, the receptor current of Ir-expressing OSNs did not adapt. In contrast, Or-expressing OSNs showed a strong Ca(2+)-dependent adaptation. The adaptation-induced changes in odor sensitivity obeyed the Weber-Fechner relation; however, surprisingly, the incremental sensitivity was reduced at low odor backgrounds but increased at high odor backgrounds. Our model for odor adaptation revealed two opposing effects of adaptation, desensitization and prevention of saturation, in dynamically adjusting odor sensitivity and extending the sensory operating range.