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In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3′ ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

Mejiro University, National Institute for Japanese Language and Linguistics, 会議名: 言語資源活用ワークショップ2021, 開催地: オンライン, 会期: 2021年9月13日-14日, 主催: 国立国語研究所 コーパス開発センター, 『現代日本語書き言葉均衡コーパス』の書籍サンプルにはNDC情報が付与されており，構築当時に情報のなかった書籍などへの増補も行われた（加藤ほか2021）。また，コーパスに付与されたNDCを利用することで，ジャンル別の特徴語の抽出などが試みられてきた（内田・藤井2015）。しかし，一般動詞など，多義的あるいは補助的に使用される語は，語義情報なしでは語彙としての分布傾向が見られにくく，ジャンル横断的な分布となる。そこで，本稿は，増補したNDC（加藤ほか前掲）を用いてジャンルの語彙分布を再確認するとともに，分類語彙表番号の付与されたBCCWJ-WLSP（加藤ほか2019）と重ね合わせることにより，語義分布に内容別の傾向が見られることを確認する。

Mejiro University, National Institute for Japanese Language and Linguistics, 会議名: 言語資源活用ワークショップ2021, 開催地: オンライン, 会期: 2021年9月13日-14日, 主催: 国立国語研究所 コーパス開発センター, コーパスに付与されたジャンル情報を用いることにより，ジャンル毎の語彙分布の傾向が確認される。しかし，レジスタによる文体差の影響や，ジャンルの分類基準の問題が考えられる。そこで，本稿は，文章内容情報が付与された文体的な影響の少ないコーパスを用い，品詞分布・語彙分布・語義分布に内容別の傾向が見られることを確認する。具体的には，『現代日本語書き言葉均衡コーパス』の新聞サブコーパス（PN，1,473サンプル）に含まれるサンプルを記事単位（5,585記事）に分割し，記事ごとの内容情報や種別情報を付与した（加藤ほか2020）データを用いる。分類語彙表番号の付与されたBCCWJ-WLSP（加藤ほか2019）と重ね合わせることにより語義分布も調査する。

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

National Institute for Japanese Language and Linguistics, Tsuda University / National Institute for Japanese Language and Linguistics, 会議名: 言語資源活用ワークショップ2019, 開催地: 国立国語研究所, 会期: 2019年9月2日−4日, 主催: 国立国語研究所 コーパス開発センター, 『現代日本語書き言葉均衡コーパス』の書籍サンプル（PB（出版）10,117サンプル・LB（図書館）10,551サンプル・OB（ベストセラー）1,390サンプル）に付与された日本十進分類法（NDC）分類記号の補助分類を拡張した。また、開発当時NDC分類記号が付与されていなかったサンプル（「分類なし」）などの見直しもあわせて行った。作業は、国立国会図書館のNDC情報を参照し、人手によって分類の確認と追加を進めた。本作業結果により、たとえば形式区分を利用し、ジャンルの分散する「随筆(-049)」「理論(-01)」「研究法(-07)」などのカテゴリでBCCWJサンプルを分類することが可能となった。このほか、時代情報や小項目が追加されたサンプルもあり、今まで以上に詳細な分類が可能となった。本発表では、情報付与作業の方法と基礎情報を報告し、分類例を示す。本作業結果データは「中納言」の検索結果として利用可能となる。

University of Tsukuba, University of Tokyo, Tsuda University / National Institute for Japanese Language and Linguistics, National Institute for Japanese Language and Linguistics/National Institute for Japanese Language and Linguistics, 会議名: 言語資源活用ワークショップ2019, 開催地: 国立国語研究所, 会期: 2019年9月2日−4日, 主催: 国立国語研究所 コーパス開発センター, 『分類語彙表』の見出し語と『岩波国語辞典第五版タグ付きコーパス2004』に含まれる国語辞典見出し語との対応表を作成した。分類語彙表は統語・意味に基づいて見出し語を分類したシソーラスであるが、その語義を規定する語釈文を含んでいない。そこで、岩波国語辞典の見出し語と対照させることで対応表を構築し、統語・意味分類と語釈文を結びつける作業を行った。作業は、見出し語表記による2部グラフを構成し、対応する見出し語対を抽出することによる。本作業は5人の作業者により平行して進めた。本作業結果により、『現代日本語書き言葉均衡コーパス』に付与された2種類の語義情報（分類語彙表番号・岩波語義タグ）との対照比較ができるようになった。本発表では、情報付与作業の方法と基礎情報を報告する。

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

The liver zonation is an important phenomenon characterized by a gradient of several functions along the liver acinus. However, this gradient remains difficult to reproduce in in-vitro conditions, making the obtention of an in-vitro method to recapitulate the liver zonation a challenging issue. In this study, we evaluated the spatial evolution of the transcriptome profile of human induced pluripotent stem cells (hiPSCs) differentiated toward hepatocytes-like cells (HLCs) phenotype in a microfluidic biochip environment. Cells collected at the inlet of the biochip, where the oxygen concentration is higher, were identified by the expression of genes involved in metabolic pathways related to cellular reorganization and cell proliferation. Cells collected in the middle and at the outlet of the biochips, where oxygen concentrations are lower, were characterized by the upregulation of genes involved in cellular detoxification processes (CYP450), PPAR signaling or arginine biosynthesis. A subset of 16 transcription factors (TFs) was extracted and identified as upstream regulators to HNF1A and PPARA. These TFs are also known as regulators to target genes engaged in the Wnt/βcatenin pathway, in the TGFβ/BMP/SMAD signaling, in the transition between epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), in the homeostasis of lipids, bile acids and carbohydrates homeostasis, in drug metabolism, in the estrogen processing and in the oxidative stress response. Overall, the analysis allowed to confirm a partial zonation-like pattern in hiPSCs-derived HLCs in the microfluidic biochip environment. These results provide important insights into the reproduction of liver zonation in-vitro for a better understanding of the phenomenon.

International audience; The differentiation of human induced pluripotent stem cells (hiPSCs) into functional hepatocytes has the potential to solve the shortage of human primary liver cells and would be of use in drug screening. In this frame, we developed a hiPSCs maturation strategy in microfluidic biochips, using a liver-on-chip approach, a promising technology mimicking in vivo physiology. Hepato-like tissues differentiated in biochips presented advanced liver features, including ALB and CYP3A4 expressing cells. The metabolomics and transcriptomics profiles of hepato-likes cells differentiated either in biochips or Petri dishes were integrated to compare their functionalities. The multi-omics analysis revealed 41 metabolites and 302 genes differentially expressed. Overall, biochip environment lead to higher degree of hepatic differentiation demonstrated by an increase in the metabolic production of lipids, fatty acids and biliary acids, which was confirmed at the transcriptome level by the modulation of expression for genes involved in related signaling pathways. This observation was correlated with higher production of fructose in biochips, together with down-regulation of genes engaged in glycolysis. In parallel, increased activity of the Krebs cycle, pentose phosphate shuffle, and fatty acid beta oxidation was observed in tissues cultured in Petri. Besides, the modulation of nitrogen metabolism was observed in transcriptomic data and confirmed by an intense production of glutamine, putrescine and creatinine and by the higher consumption of spermidine measured in Petri.

National Institute for Japanese Language and Linguistics, Nippon Institute of Technology, Tsuda University, 会議名: 言語資源活用ワークショップ2018, 開催地: 国立国語研究所, 会期: 2018年9月4日-5日, 主催: 国立国語研究所 コーパス開発センター, 現在，『現代日本語書き言葉均衡コーパス』に含まれる書籍サンプルに付与された日本十進分類法（NDC）分類記号に，補助分類を拡張する作業を進めている。国立国会図書館のNDC情報（8版・9版）を参照し，人手によって補助分類の確認と追加を行う。本発表は，現在までに作業の完了した図書館サブコーパス10,551サンプルについて，情報付与作業方法とその結果を報告する。本作業により，たとえば形式区分を利用し，ジャンルの分散する「随筆」「理論」「研究法」などのカテゴリでBCCWJサンプルを分類することが可能となる。そこで，付与した形式区分「随筆」サンプル群を例とし，語彙特徴から文体的な傾向を調査した。さらに，柏野（2015）の文体指標を用い，「随筆」の文体特徴として考えられてきた「主観的」で「軟らかく」「くだけている」傾向などを確認する。

Published single-cell datasets are rich resources for investigators who want to address questions not originally asked by the creators of the datasets. The single-cell datasets might be obtained by different protocols and diverse analysis strategies. The main challenge in utilizing such single-cell data is how we can make the various large-scale datasets to be comparable and reusable in a different context. To challenge this issue, we developed the single-cell centric database ‘SCPortalen’ (http://single-cell.clst.riken.jp/). The current version of the database covers human and mouse single-cell transcriptomics datasets that are publicly available from the INSDC sites. The original metadata was manually curated and single-cell samples were annotated with standard ontology terms. Following that, common quality assessment procedures were conducted to check the quality of the raw sequence. Furthermore, primary data processing of the raw data followed by advanced analyses and interpretation have been performed from scratch using our pipeline. In addition to the transcriptomics data, SCPortalen provides access to single-cell image files whenever available. The target users of SCPortalen are all researchers interested in specific cell types or population heterogeneity. Through the web interface of SCPortalen users are easily able to search, explore and download the single-cell datasets of their interests.

会議名: 言語資源活用ワークショップ2017, 開催地: 国立国語研究所, 会期: 2017年9月5日-6日, 主催: 国立国語研究所 コーパス開発センター, 比喩表現類型に意味情報を付与し，意味的な要素の結合を比喩表現の指標とした用例収集を試みた。まず，日本語比喩表現の類型を『分類語彙表増補改訂版』に基づく意味分類で整理した。具体的には，国立国語研究所報告57『比喩表現の理論と分類』に挙げられた結合類型（5,537種類）に含まれる要素の自立語すべてに人手で分類語彙表番号を付与し，比喩表現類型を意味分類において整理したデータを作成した。次に，意味分類の結合を比喩表現の指標として，分類語彙表番号を付与した「現代日本語書き言葉均衡コーパス」のコアデータの一部（約19万短単位）を調査した。本発表では，本手法によって取得できたBCCWJの比喩表現例を示すとともに，意味分類結合としての日本語比喩表現の使用傾向を確かめる。

会議名: 言語資源活用ワークショップ2016, 開催地: 国立国語研究所, 会期: 2017年3月7日-8日, 主催: 国立国語研究所 コーパス開発センター, 『現代日本語書き言葉均衡コーパス（BCCWJ）』に分類語彙表番号を付与する作業を開始した。『分類語彙表増補改訂版』（2004）の分類語彙表番号を，短単位と長単位のそれぞれにアノテーションする。作業にあたり，人手でUniDic語彙素IDに対応させたデータ（近藤・田中，2017）を用い，該当可能性のある番号を列挙する。作業者は，該当する意味分類が選択可能であれば選択し，選択できない場合や対応のない場合には，新たに適切な番号を付与する。本発表では，番号付与作業基準と作業状況，作業結果を用いた調査例を報告する。

会議名: 言語資源活用ワークショップ2016, 開催地: 国立国語研究所, 会期: 2017年3月7日-8日, 主催: 国立国語研究所 コーパス開発センター, 日本語は冠詞のない言語である．ゆえに，日本語から冠詞を持つ言語への翻訳の際には，人によるものでも，機械によるものでも冠詞選択の問題を引き起こすことになる．冠詞選択には，ソースとなる言語における名詞句の情報構造(定性，特定性など) が影響を与える．本稿では，翻訳における冠詞選択の問題を軽減させるため，『現代日本語書き言葉均衡コーパス』のテキスト(新聞(PN) コアデータ16 サンプル) 内の名詞句に対し，情報構造に関係する文法情報のタグをアノテーションした結果を報告する．特に，本稿ではそのアノテーションの概要と基礎統計について述べる．

Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

International audience; We investigated the human induced pluripotent stem cells (hiPSCs) during a sequential in vitro step-by-step differentiation into hepatocyte-like cells (HLCs) using nanoCAGE, a method for promoters, transcription factors, and transcriptome analysis. Specific gene clusters reflected the different steps of the hepatic differentiation. The proliferation step was characterized by a typical cell cycle and DNA replication. The hepatic endoderm and the HLC steps were marked by a common signature including cell interactions with extracellular matrix (ECM), lipoproteins and hepatic biomarkers (such as albumin and alpha-fetoprotein). The specific HLC profile was characterized by important transcription factors such as HIF1A, JUN, MAF, KLF6, BMP4 and with a larger expression of genes related to Wnt signaling, extracellular matrix, lipid metabolism, urea cycle, drugs, and solute transporters. HLC profile was also characterized by the activation of upstream regulators such as HNF1A, MEIS2, NFIX, WRNIP1, SP4, TAL1. Their regulatory networks highlighted HNF4a as a bridge and linked them to important processes such as EMT–MET transitions, ECM remodeling and liver development pathways (HNF3, PPARA signaling, iron metabolism) along the different steps of differentiation.

International audience; Human hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC) may provide an unlimited supply of cells for in vitro liver models. However, hiPSC differentiation remains a major challenge due to immaturity of the hepatocytes obtained and the high cost of differentiation protocols currently proposed. Here, we studied the efficacy of new protocol, with reduction of growth factors, for the generation of hepatocyte-like cells from hiPSC. We performed metabolomic and mRNA analysis by RTqPCR and nanoCAGE processing to identify and understand key metabolisms during differentiation. By reducing the change frequency of the culture medium in the new protocol, we successfully generated hepatocyte-like cells producing albumin, urea, and CYP3A4 positive. The metabolomic analysis successfully extracted both signatures, common and specific, for each differentiation step. Integrating the metabolomic data with transcriptomic contributed to explaining the kinetics of carbohydrate, lipid and nitrogen metabolism throughout differentiation. The information extracted during differentiation showed that the cells moved from an aerobic-like respiration pattern to a mitochondrial oxidative respiration pattern in both protocols. Reducing culture medium renewal led to reduced glucose consumption, followed by fructose production and significant extracellular lipogenesis throughout differentiation. We believe that the overall dataset can provide information on the sequence of process.

The development of complex, multi-step methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where an optimum is sought in a multidimensional parameter space through step-by-step optimisations. The difficulty of miniaturising reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates more than 600 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterisation of biochemical systems.

In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the ɑ-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.

International audience; Human induced pluripotent stem (hiPS) cells represent a potential source of human cells for regenerative and personalized medicine applications. It is envisioned that hiPS cells could be an efficient tool for disease modelling and drug therapy testings. However, the successful derivation of hiPS cells to mature hepatocytes remains a challenge as far as the current proposed protocols lead to immature patterns and are very expensive (due to growth factors cost). In this work, we have compared two protocols of differentiation to produce hepatocyte-like cells. The first protocol is the conventional protocol adapted from Si-Tayeb et al. in 2010. By changing the frequency of culture medium exchange, we reduced the use of growth factors in the second protocol by 40%. Both protocols lead to hepatocyte-like characteristics including albumin (ALB) and cytochrome P450/3A4 (CYP3A4) expression in cells. Albumin production reached 2500 ng/mL/day for both protocols. The transcription factor motif activity analysis via transcriptomic data obtained with the Cap Analysis Gene Expression (CAGE) technology confirmed a typical hepatic profile for both protocols (such as activation of HNF1 A and HNF1B promoters). However, specific patterns were also detected with higher mRNA levels of WT1, MTF1, HCFC1, and PGCα1 in protocol 2. The overall transcriptomic analysis also revealed a modulation of targets involved in glucose homeostasis, in cell remodeling, lipid pathways and chromatin processing. However, the alpha-fetoprotein (AFP) production remained very high and the function of CYP3A4 was weak in both protocols illustrating still an immature profile in both protocols.

Transcription of human papillomavirus (HPV) genes proceeds unidirectionally from multiple promoters. Direct profiling of transcription start sites (TSSs) by Cap Analysis Gene Expression (CAGE) is a powerful strategy for examining individual HPV promoter activity. The objective of this study was to evaluate alterations of viral promoter activity during infection using CAGE technology. We used CAGE-based sequencing of 46 primary cervical samples, and quantitatively evaluated TSS patterns in the HPV transcriptome at a single-nucleotide resolution. TSS patterns were classified into two types: early promoter-dominant type (Type A) and late promoter-dominant type (Type B). The Type B pattern was more frequently found in CIN1 and CIN2 lesions than in CIN3 and cancer samples. We detected transcriptomes from multiple HPV types in five samples. Interestingly, in each sample, the TSS patterns of both HPV types were the same. The viral gene expression pattern was determined by the differentiation status of the epithelial cells, regardless of HPV type. We performed unbiased analyses of TSSs across the HPV genome in clinical samples. Visualising TSS pattern dynamics, including TSS shifts, provides new insights into how HPV infection status relates to disease state.

The development of complex methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where optima are sought in a multidimensional parameter space through step-by-step optimizations. The difficulty of miniaturizing reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates 648 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterization of biochemical systems.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization., Single-cell transcriptomic profiling allows the exploration of cellular heterogeneity but commonly focuses on the 3′-end of the transcript. Here the authors introduce C1 CAGE, which detects the 5′ transcript end in a multiplexed microfluidic system.

The Japanese language is written without the use of articles. Therefore, when translating from Japanese to languages with articles by either humans or machines, issues in article selection arise, which are affected by the information structure (definiteness, specificity, and others) of the source language. This paper presents the annotation data of the information structure (information status, commonness, definiteness, specificity, animacy, sentience, agentivity) of the “Balanced Corpus of Contemporary Written Japanese,” to address article selection issues in translation. We present the annotation schema and statistics. Evaluation using the Kappa value demonstrates that there is a correspondence between the information status, commonness, definiteness, and specificity. Thus, we conclude that these grammatical labels affect article selection.

Transcripts in all eukaryotes are characterized by the 5'-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5'-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5'-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.

Transcripts in all eukaryotes are characterized by the 5'-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5'-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5'-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.

(ProQuest: ... denotes non-USASCII text omitted.)INTRODUCTIONIn December 2011, the National Institute for Japanese Language and Linguistics (NINJAL) released a 100-million-word balanced corpus - the Balanced Corpus of Contemporary Written Japanese (BCCWJ) (Maekawa et al., 2013) - which was compiled from 2006 through 2011. The BCCWJ includes a wide range of textual registers, styles and genres, which ensures a balance and representativeness of coverage. Its samples were collected from rigidly defined statistical populations by random sampling. However, the BCCWJ does not include low-frequency linguistic phenomena, as do the web-scale corpus-based studies in recent advanced corpus linguistics, because BCCWJ's scale is smaller. To address this issue, in 2011 NINJAL launched another corpus compilation project that is intended to construct a ten-billion-word web corpus by 2016. In this paper, we describe the basic design of the main categories included in the construction of this web-scale Japanese linguistic corpus.We present an overview of the design of the online language resources in terms of the four main categories: Page Collection, Linguistic Annotation, Release, and Preservation. For Page Collection, web crawlers are employed to collect web text by crawling 100 million pages every three months and retaining several versions for three-month periods. For Linguistic Annotation, the linguistic studies web corpus contains annotated linguistic information. To improve the usability of these linguistic resources, normalization is performed, such as removal of tags, as well as word segmentation, dependency parsing, and register estimation. For Release, word lists and n-gram data based on the crawled and annotated text corpus are published. In addition, applications are being developed that enable searching for morphosyntax patterns in the tenbillion-word corpus. For Preservation, the crawled pages are preserved in chronological order as web archives primarily to support the survey of ongoing linguistic changes.Frequently used fixed phrases from copy sites or pages tend to appear in the web corpus. The existence of these sites and pages prompts us to evaluate web corpus statistics; moreover, to alleviate the issue, we introduce deduplication of sentences, which makes the web corpus more useful for linguistic research due to elimination of fixed expressions on the web.By the end of 2013, the project reflected stable progress for page collection and linguistic annotation. Below, we present the design of the four categories and current status of the corpus with statistics. In 'Related Work', we outline other work concerning web-scale Japanese language resources. In 'Web Corpus Design Overview', we present the design of the four categories. In 'Statistics', we discuss the current project status in terms of page collection and linguistic annotation with the use of statistics. In addition, we evaluate the effects of deduplication of sentences. Our conclusions and reference to future work are provided in 'Conclusions'.RELATED WORKSeveral web-scale Japanese language resources have already been compiled and publicly released by search engine providers and portal site companies. Most of the resources are simply word lists and/or n-gram statistics primarily because of constraints of Japanese copyright laws.Google released the Japanese Web N-gram Version 1 (Google N-gram) - i.e. word n-grams and their observed frequency counts generated from more than 225 billion words of text, which were crawled during July 2007. Baidu released two versions of its Baidu Japan Corpus. One is the Baidu Blog and Forum-Timed Corpus, which comprises text from blogs and bulletin boards/forums that were crawled between 2000 and 2010. The samples are divided by months, and ten million sentences were randomly selected. The other is the Baidu Mobile Web Corpus with Emoji, which comprises texts crawled by mobile search engines. …

Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions, and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons pose a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome—the translatome—from selected subcellular domains of specific neurons, and apply it to the Purkinje cells (PCs) in the rat cerebellum. We combined microdissection, translating ribosome affinity purification (TRAP) in nontransgenic animals, and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)–associated ribosomes in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities.

We used a transgenic HeLa cell line that reports cell cycle phases through fluorescent, ubiquitination-based cell cycle indicators (Fucci), to produce a reference dataset of more than 270 curated single cells. Microscopic images were taken from each cell followed by RNA-sequencing, so that single-cell expression data is associated to the fluorescence intensity of the Fucci probes in the same cell. We developed an open data management and quality control workflow that enables users to replicate the processing of the sequence and microscopic image data that we deposited in public repositories. The workflow outputs a table with metadata, that is the starting point for further studies on these data. Beyond its use for cell cycle studies, We also expect that our workflow can be adapted to other single-cell projects using a similar combination of sequencing data and fluorescence measurements.

Transcriptome studies based on quantitative sequencing estimate gene expression levels by measuring the abundance of target RNAs in libraries of sequence reads. The sequencing cost is proportional to the total number of sequenced reads. Therefore, in order to cover rare RNAs, considerable quantities of abundant and identical reads have to be sequenced. This major limitation can be lifted by strategies used to deplete the library from some of the most abundant sequences. However, these strategies involve either an extra handling of the input RNA sample, or the use of a large number of reverse-transcription primers (termed "not-so-random primers"), which are costly to synthetize and customize. Here, we demonstrate that with a precise selection of only 40 "pseudo-random" reverse-transcription primers, it is possible to decrease the rate of undesirable abundant sequences within a library without affecting the transcriptome diversity. "Pseudo-random" primers are simple to design, and therefore are a flexible tool for enriching transcriptome libraries in rare transcripts sequences.

Cholecystokinin (CCK) plays a major role in the regulation of pancreatic enzyme secretion based on its binding to the CCK-A receptor (CCK-AR). While CCK-AR is known to be expressed in rat islet B cells, the localization of CCK-AR in rat pancreatic A and D cells remains poorly understood. The aim of this study was to identify the localization of CCK-AR in rat pancreatic islets by means of double immunofluorescence straining with antibodies against CCK-AR, glucagon, insulin and somatostatin and with in situ hybridization to detect its transcript. CCK-AR-like immunoreactive cells were found to overlap both with glucagon-like immunoreactive cells and insulin-like immunoreactive cells but not with somatostatin-like immunoreactive cells. An in situ hybridization study using a cRNA probe for CCK-AR revealed that CCK-AR mRNA was expressed in the center and periphery of the pancreatic islets. Further to this, immunofluorecsence staining using anti-glucagon antibody was carried out after in situ hybridization using the CCK-AR cRNA probe in order to identify CCK-AR mRNA expressing cells. CCK-AR mRNA exhibited a distribution pattern almost identical to that of glucagon-like immunoreactive cells. These results show clearly that CCK-AR exists not only in B but also in A cells of the rat pancreas, suggesting that CCK regulates the secretion of insulin and glucagon at least partly via CCK-AR.