limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.

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limma powers differential expression analyses for RNA-sequencing and microarray studies

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

/pdf/mapping-and-quantifying-mammalian-transcriptomes-by-rna-seq-1egh5oivst.pdf

Mapping and quantifying mammalian transcriptomes by RNA-Seq.

RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

/pdf/rna-seq-a-revolutionary-tool-for-transcriptomics-bj1lajhogb.pdf

RNA-Seq: a revolutionary tool for transcriptomics

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

/pdf/initial-sequencing-and-comparative-analysis-of-the-mouse-5a9n62pifj.pdf

Initial sequencing and comparative analysis of the mouse genome.

Warburg first reported that tumours are characterized by abnormally increased glucose metabolism accompanied by increase production of lactate. This is a basic principle underlying cancer detection by the glucose analogue 18-F-fluoro-2-deoxy-D-glucose (FDG). FDG positron emission tomography (PET) is currently used widely to examine virtually any part of the body in order to detect tumours, e.g., lung, breast, colorectal, pancreatic and head and neck cancers, malignant lymphoma and malignant melanoma. The advantage of whole-body FDG-PET in comparison with the other imaging modalities is that it allows the entire body to be surveyed seamlessly within a reasonably short period. Furthermore, the staging of most cancers can be determined. The characteristics of whole-body FDG-PET seem to satisfy the requirements for cancer screening. PET used simultaneously with conventional tests can prevent the overlooking of cancer, reduce false-positive results and assist in the interpretation of CT and MRI images. Thus, PET can play a supportive role when used with conventional screening tests. In 1994, PET was applied to cancer screening for the first time at our Imaging Center at Lake Yamanaka in Japan. Within 12 years after starting, a total of 10,292 asymptomatic individuals (6,227 men and 4,065 women; mean age, 52.2 and 52.9 years) participated in 29,090 screening sessions. As a result, malignant tumours were demonstrated in 355 of the 10,292 participants (2.61%). PET findings were true-positive in 175 of the 355 cancers (49.3%).

[Cancer screening with PET: advantages and limitations].

S-1 is a recently developed agent that reduces the gastrointestinal toxicity of 5-fluorouracil without affecting its antitumor activity. We encountered a patient with advanced gastric cancer, who responded to S-1 monochemotherapy and has maintained complete remission for over 4 years. The case was of a 61-year-old man who presented with abdominal pain in July 2001 and was diagnosed with stage IV gastric cancer (T4N2M0). Curative surgery such as gastrectomy was not appropriate, and mono-chemotherapy with S-1 was administered. This was given for 4 consecutive weeks at a dose of 120 mg/day, followed by a 2-week rest period; 18 courses were administered until September 2003. These cases suggest that a subgroup of patients with advanced gastric cancer may attain a complete response with S-1 chemotherapy, with or without gastrectomy.

/pdf/a-case-of-stage-iv-gastric-cancer-long-term-remission-r0j3k65z48.pdf

A Case of Stage IV Gastric Cancer: Long-term remission achieved with S-1 mono-chemotherapy

Heterogeneous phenotypes of cancer cells enable them to adapt to various environments. The heterogeneity results from diversity of genome, transcriptome, and epigenome at a single-cell level. The C1 system can automatically perform single-cell capture and whole genome amplification (WGA) or whole transcription amplification (WTA) by MDA or Smart-Seq, respectively. Here, we describe the protocols for WGA and WTA from a single cell by using the C1 system and the protocols for sequence library preparation from amplified gDNA and cDNA. We also described about the computational analysis for single-cell data of cancer.

Single-Cell DNA-Seq and RNA-Seq in Cancer Using the C1 System

[Problem] To provide an apparatus and a method for inhibiting the growth of algae, whereby it becomes possible to effectively inhibit the growth of algae in a closed water area using a microorganism. [Solution] An apparatus for inhibiting the growth of algae according to the present invention is equipped with: a tubular reaction vessel which has, formed at the bottom part thereof, a gas inlet part through which an oxygen-containing gas can be introduced and a water inlet part through which water can be introduced, and also has, formed at the top part thereof, a gas discharge part through which at least the gas can be discharged; a degassing tube in which one end side thereof is connected to the gas discharge part so that the gas discharged through the gas discharge part can pass through the inside of the tube and the other end side thereof extends so as to protrude through water surface; and a microorganism carrier which is placed in the reaction vessel and can carry a microorganism capable of oxidizing a metal that is required for the growth of the algae. In the apparatus, the reaction vessel and/or the above-mentioned one end side of the degassing tube is provided with a water flow direction control part by which the direction of the vertically upward water flow, which is generated by the flow of the gas floating in the reaction vessel, can be inclined from vertically above so that the water flow can be discharged into the water area.

Apparatus and method for inhibiting growth of algae

RNA-seq using long-read sequencing, such as nanopore and SMRT (Single Molecule, Real-Time) sequencing, enabled the identification of the full-length structure of RNA molecules. Several tools for long-read RNA-seq were developed recently. In this section, we introduce an analytical pipeline of long-read RNA-seq for isoform identification and the estimation of expression levels using minimap2, TranscriptClean, and TALON. We applied this pipeline to the public direct RNA-seq data of the HAP1 and HEK293 cell lines to identify transcript isoforms which can be detected only using long-read RNA-seq data.

Yutaka Suzuki

Papers

[Cancer screening with PET: advantages and limitations].

A Case of Stage IV Gastric Cancer: Long-term remission achieved with S-1 mono-chemotherapy

Single-Cell DNA-Seq and RNA-Seq in Cancer Using the C1 System

Apparatus and method for inhibiting growth of algae

Transcript Identification Through Long-Read Sequencing.