Biology subjects

Sugino, K.

Publications and source records attributed to Sugino, K..

9 recordsLinked to original sources

bioRxiv2026

The Phantom of the PCR: detection and consequences of spurious UMIs in mainstream RNA sequencing

Unique molecular identifiers (UMIs) support digital molecular counting by tagging molecules before amplification, but assume that UMIs are incorporated only during reverse transcription. Residual UMI-bearing oligonucleotides can instead reprime during preamplification PCR, creating "phantom" UMIs on genuine cDNA that inflate counts and evade standard deduplication. We model phantom generation as a two-state branching process and show that it produces a heavy-tailed reads-per-UMI distribution distinct from that of true UMIs. Using this signature, PhantomUMI detects and estimates contamination from clone-size distributions, subject to a coverage-dependent identifiability limit. Across 23 datasets spanning published studies and companion experiments, we find signatures consistent with phantom-UMI generation, including in current 10x GEM-X chemistry. Simulations show that phantoms inflate molecule counts and distort fold-changes. Model-based correction removes average count inflation but does not recover the distorted fold-changes, indicating that phantom UMIs are best prevented experimentally, as implemented in the companion Omega-seq method.

bioinformatics↗

bioRxiv2026

ESPeR-seq: Extremely Sensitive and Pure, End-to-end, RNA-seq library preparation

PCR-based RNA sequencing methods generate phantom unique molecular identifiers (UMIs) when residual UMI-bearing oligonucleotides reprime during amplification, inflating molecule counts and corrupting relative abundances. They also incur non-specific amplification that limits sensitivity, while synthetic poly-T tracts disrupt sequencing and obscure transcript 3' ends. Here we present Omega-seq, which restricts UMI incorporation to reverse transcription using uracil-containing, USER-excisable template-switching oligonucleotides and a uracil-intolerant polymerase. Omega-seq produces near-background-free libraries, distinguishes 10 fg of input RNA from negative controls, and eliminates bead cleanup. An Omega-shaped dT primer prevents poly-T-induced dephasing, enabling stranded, nucleotide-resolution mapping of transcript ends. Dilution series, ERCC spike-ins, and a rarefaction metric show that Omega-seq has the lowest UMI inflation among methods tested. In individual Drosophila neuroblasts, phantom-UMI inflation remains minimal; pooled neuroblast and neuron coverage enables de novo discovery of novel multi-exon genes, 3' UTR extensions, and candidate enhancer RNAs.

genomics↗

bioRxiv2020

Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling

Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.

developmental biology↗

bioRxiv2020

Three-dimensional understanding of the morphological complexity of the human uterine endometrium

SummaryThe histological basis of the human uterine endometrium has been established by 2D observation. However, the fundamental morphology of endometrial glands is not sufficiently understood because these glands have complicated winding and branching patterns. To construct a big picture of endometrial gland structure, we performed tissue-clearing-based 3D imaging of human uterine endometrial tissue. Our 3D immunohistochemistry and 3D layer analyses revealed that endometrial glands formed a plexus network in the stratum basalis, similar to the rhizome of grass. We then extended our method to assess the 3D morphology of adenomyosis, a representative “endometrium-related disease”, and observed 3D morphological features including direct invasion of endometrial glands into the myometrium and an ant colony-like network of ectopic endometrial glands within the myometrium. Thus, 3D analysis of the human endometrium and endometrium-related diseases will be a promising approach to better understand the pathologic physiology of the human endometrium.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2019

CAMIO for deletion analysis of endogenous DNA sequences in multicellular organisms

The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve high-throughput analysis of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5UTR sequences crucial for chinmos post-transcriptional regulation.

genomics↗

bioRxiv2019

Robust Reconstruction of CRISPR and Tumor Lineage Using Depth Metrics

Lineage reconstruction using CRISPR edited barcodes are becoming wide-spread and methods robust against noise are in need. Neighbor-Joining (NJ) algorithm is a robust distance based algorithm extensively used in phylogeny field. NJ is also used for CRISPR-encoded-lineage (CEL) reconstruction with proper re-rooting since NJ is unrooted algorithm. However, we found NJ works without re-rooting for reconstructing CEL when the lineage contains multiple trees but not for a single tree. Examining why this is the case leads to the idea of \"depth metrics\". The notion of depth metrics also naturally explains why Russell-Rao metric, previously found best metric for CEL reconstruction, works well. Furthermore, based on the probabilistic model of CEL, we constructed a new metric that performs better than Russell-Rao metric. We also propose inferring ancestral code during reconstruction instead of using a linkage method. These, together with Nearest-Neighbor-Interchange resulted in a new robust method for reconstructing CEL or tumor-cell-lineages which share same assumptions as CEL.

developmental biology↗

bioRxiv2019

Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

Delineating cell lineages is a prerequisite for understanding the genesis of cell types. Recent studies have demonstrated the feasibility of generating and reconstructing CRISPR/Cas9-coded cell lineages. However, these works have not investigated the limitations or optimality of the encoding or reconstruction processes. Here, we surveyed a multitude of reconstruction algorithms and found hierarchical clustering, with a metric based on the number of shared Cas9 edits, provides the best reconstruction. As to the efficiency, the simple encoding method, with constant Cas9/gRNA edit rate, produces exponential reduction in available coding units and severely limits the trackable depth of lineages. To overcome this, we propose alternative encoding methods, one based on parallel gRNA cascades enabled by CLADES, and another based on variable Cas9 editing rate. Both significantly increase the trackable depth. In summary, we provide a theoretical basis in understanding, designing and analyzing efficient and robust CRISPR-based cell lineage tracking system.

developmental biology↗

bioRxiv2017

The Transcriptional Logic of Mammalian Neuronal Diversity

The mammalian nervous system is constructed of many cell types, but the principles underlying this diversity are poorly understood. To assess brain-wide transcriptional diversity, we sequenced the transcriptomes of the largest collection of genetically and anatomically identified neuronal classes. Using improved expression metrics that distinguish information content from signal-to-noise-ratio, we found that homeobox transcription factors contain the highest information about cell types and have the lowest noise. Genes that contribute the most to neuronal diversity tend to be long and enriched in factors specifically involved in neuronal function. Genome accessibility measurements reveal that long genes have more candidate regulatory elements arrayed in more distinct patterns. These elements frequently overlap interspersed repeats (mobile elements) and the pattern of repeats is predictive of gene expression. New regulatory sites resulting from elongation of neuronal genes by mobile elements may be an evolutionary force enhancing nervous system complexity.

neuroscience↗

bioRxiv2017

Stem cell intrinsic, Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors

Drosophila type II neuroblasts produce numerous neurons and glia due to transiently amplifying, intermediate neural progenitors (INP). Consecutively born INPs produce morphologically distinct progeny, presumably due to temporal patterning in type II neuroblasts. We therefore profiled type II neuroblasts transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining Imp expression throughout brain development expands the number of neurons/glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Further, we reveal that the transcription factor, Seven-up initiates progression of the Imp/Syp gradients. Interestingly, genetic manipulations that fix Imp or Syp levels still yield progeny with a small range of early fates. We propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

neuroscience↗