Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations  Recently  application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease  Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases  Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts  We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease  We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing  We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL  This study further improves our understanding of the processes and pathways involved in lymphomagenesis  and some of the pathways mutated here may indicate new avenues for therapeutic intervention 
                Discoveries from cancer genome sequencing have the potential to translate into advances in cancer prevention  diagnostics  prognostics  treatment and basic biology  Given the diversity of downstream applications  cancer genome-sequencing studies need to be designed to best fulfil specific aims  Knowledge of second-generation cancer genome-sequencing study design also facilitates assessment of the validity and importance of the rapidly growing number of published studies  In this Review  we focus on the practical application of second-generation sequencing technology (also known as next-generation sequencing) to cancer genomics and discuss how aspects of study design and methodological considerations - such as the size and composition of the discovery cohort - can be tailored to serve specific research aims 
                Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%  Previously  we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples  To explore the effects of FOXO1 mutations  we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines  FOXO1 mutations were found in 8 6% (24/279) of DLBCL cases: 92 3% (24/26) of mutations were in the first exon  46 2% (12/26) were recurrent mutations affecting the N-terminal region  and another 38 5% (10/26) affected the Forkhead DNA binding domain  Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation  loss of interaction with 14-3-3  and nuclear retention  FOXO1 mutation was associated with decreased overall survival in patients treated with rituximab  cyclophosphamide  doxorubicin  vincristine  and prednisone (P =  037)  independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI)  This association was particularly evident (P =  003) in patients in the low-risk R-IPI categories  The independent relationship of mutations in FOXO1 to survival  transcending the prognostic influence of the R-IPI and COO  indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis 
                The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer  The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells  Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment  We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors  We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived  A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells  The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line  We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors  The observed mutations  aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer 
                Networks are typically visualized with force-based or spectral layouts  These algorithms lack reproducibility and perceptual uniformity because they do not use a node coordinate system  The layouts can be difficult to interpret and are unsuitable for assessing differences in networks  To address these issues  we introduce hive plots (http://www hiveplot com) for generating informative  quantitative and comparable network layouts  Hive plots depict network structure transparently  are simple to understand and can be easily tuned to identify patterns of interest  The method is computationally straightforward  scales well and is amenable to a plugin for existing tools 
                Oligodendroglioma is characterized by unique clinical  pathological  and genetic features  Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited  We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution  As anticipated  there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16)  In addition  we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13 2 in 13/16 tumours  These discovery set mutations were validated by deep sequencing of 13 additional tumours  which revealed seven others with CIC mutations  thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%)  In contrast  deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60  2%)  Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas  nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain  The remaining nine were found in other exons and frequently included truncations  CIC mutations were highly associated with oligodendroglioma histology  1p/19q co-deletion  and IDH1/2 mutation (p &lt  0 001)  Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours  in a background of 1p/19q co-deletion  hemizygous CIC mutations are likely important  We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation  Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation  IDH1/2 mutation  and 1p/19q co-deletion 
                Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs)  Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL  We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations  and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations  Genes with roles in histone modification were frequent targets of somatic mutation  For example  32% of DLBCL and 89% of FL cases had somatic mutations in MLL2  which encodes a histone methyltransferase  and 11 4% and 13 4% of DLBCL and FL cases  respectively  had mutations in MEF2B  a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones  Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis 
                To analyze the relationship between antisense transcription and alternative splicing  we developed a computational approach for the detection of antisense-correlated exon splicing events using Affymetrix exon array data  Our analysis of expression data from 176 lymphoblastoid cell lines revealed that the majority of expressed sense-antisense genes exhibited alternative splicing events that were correlated to the expression of the antisense gene  Most of these events occurred in areas of sense-antisense (SAS) gene overlap  which were significantly enriched in both exons and nucleosome occupancy levels relative to nonoverlapping regions of the same genes  Nucleosome occupancy was highly correlated with Pol II abundance across overlapping regions and with concomitant increases in local alternative exon usage  These results are consistent with an antisense transcription-mediated mechanism of splicing regulation in normal human cells  A comparison of the prevalence of antisense-correlated splicing events between individuals of Mormon versus African descent revealed population-specific events that may indicate the continued evolution of new SAS loci  Furthermore  the presence of antisense transcription was correlated to alternative splicing across multiple metazoan species  suggesting that it may be a conserved mechanism contributing to splicing regulation 
                Next generation sequencing has brought epigenomic studies to the forefront of current research  The power of massively parallel sequencing coupled to innovative molecular and computational techniques has allowed researchers to profile the epigenome at resolutions that were unimaginable only a few years ago  With early proof of concept studies published  the field is now moving into the next phase where the importance of method standardization and rigorous quality control are becoming paramount  In this review we will describe methodologies that have been developed to profile the epigenome using next generation sequencing platforms  We will discuss these in terms of library preparation  sequence platforms and analysis techniques 
                In alternative expression analysis by sequencing (ALEXA-seq)  we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues  As proof of principle  we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines  We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR  quantitative PCR and Sanger sequencing  We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping  alternative splice site usage and intron retention  Alternative expression annotation databases  source code  a data viewer and other resources to facilitate analysis are available at http://www alexaplatform org/alexa_seq/ 
                Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells  Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype  but thus far few GCB-specific mutations have been identified  Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2  which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27)  After the recent discovery of mutations in KDM6A (UTX)  which encodes the histone H3K27me3 demethylase UTX  in several cancer types  EZH2 is the second histone methyltransferase gene found to be mutated in cancer  These mutations  which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641)  occur in 21 7% of GCB DLBCLs and 7 2% of FLs and are absent from ABC DLBCLs  Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro 
                Transcriptome analysis has been a key area of biological inquiry for decades  Over the years  research in the field has progressed from candidate gene-based detection of RNAs using Northern blotting to high-throughput expression profiling driven by the advent of microarrays  Next-generation sequencing technologies have revolutionized transcriptomics by providing opportunities for multidimensional examinations of cellular transcriptomes in which high-throughput expression data are obtained at a single-base resolution 
                We created a visualization tool called Circos to facilitate the identification and analysis of similarities and differences arising from comparisons of genomes  Our tool is effective in displaying variation in genome structure and  generally  any other kind of positional relationships between genomic intervals  Such data are routinely produced by sequence alignments  hybridization arrays  genome mapping  and genotyping studies  Circos uses a circular ideogram layout to facilitate the display of relationships between pairs of positions by the use of ribbons  which encode the position  size  and orientation of related genomic elements  Circos is capable of displaying data as scatter  line  and histogram plots  heat maps  tiles  connectors  and text  Bitmap or vector images can be created from GFF-style data inputs and hierarchical configuration files  which can be easily generated by automated tools  making Circos suitable for rapid deployment in data analysis and reporting pipelines 
                We describe a new method  Tag-seq  which employs ultra high-throughput sequencing of 21 base pair cDNA tags for sensitive and cost-effective gene expression profiling  We compared Tag-seq data to LongSAGE data and observed improved representation of several classes of rare transcripts  including transcription factors  antisense transcripts  and intronic sequences  the latter possibly representing novel exons or genes  We observed increases in the diversity  abundance  and dynamic range of such rare transcripts and took advantage of the greater dynamic range of expression to identify  in cancers and normal libraries  altered expression ratios of alternative transcript isoforms  The strand-specific information of Tag-seq reads further allowed us to detect altered expression ratios of sense and antisense (S-AS) transcripts between cancer and normal libraries  S-AS transcripts were enriched in known cancer genes  while transcript isoforms were enriched in miRNA targeting sites  We found that transcript abundance had a stronger GC-bias in LongSAGE than Tag-seq  such that AT-rich tags were less abundant than GC-rich tags in LongSAGE  Tag-seq also performed better in gene discovery  identifying &gt 98% of genes detected by LongSAGE and profiling a distinct subset of the transcriptome characterized by AT-rich genes  which was expressed at levels below those detectable by LongSAGE  Overall  Tag-seq is sensitive to rare transcripts  has less sequence composition bias relative to LongSAGE  and allows differential expression analysis for a greater range of transcripts  including transcripts encoding important regulatory molecules 
                Diagnosing constitutional pathogenic copy number variants requires measuring the submicroscopic segmental chromosomal imbalance  The Affymetrix GeneChip mapping array has been used to measure duplication and deletion of genetic material in DNA samples  This protocol describes the measurement and analysis processes  specifically the computational analyses that are involved in identifying pathogenic copy number variants 
                Changes to covalent modifications of DNA and histones can be induced via environmental stimuli such as nutrients  hormones and drugs  These changes can be both transient and heritable in nature and provide a framework in which to investigate how environment and lifestyle choices impact disease susceptibility and progression  Furthermore  these modifications are central to chromatin dynamics and  as such  play key roles in many biological processes involving chromatin  such as DNA replication and repair  transcription and development  In this review we provide an overview of recent advances in our understanding of the roles that DNA and histone modification play in the onset and progression of human disease 
                A new generation of sequencing technologies  from Illumina/Solexa  ABI/SOLiD  454/Roche  and Helicos  has provided unprecedented opportunities for high-throughput functional genomic research  To date  these technologies have been applied in a variety of contexts  including whole-genome sequencing  targeted resequencing  discovery of transcription factor binding sites  and noncoding RNA expression profiling  This review discusses applications of next-generation sequencing technologies in functional genomics research and highlights the transforming potential these technologies offer 
                Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease  Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material  To facilitate analyses of such samples  whole genome amplification (WGA) techniques were developed  In this study  we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays  We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform  We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants  Our analysis indicates that many artifacts are reproducible  and may correlate with proximity to chromosome ends and GC content  Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs  Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples 
                Genome rearrangements have long been recognized as hallmarks of human tumors and have been used to diagnose cancer  Techniques used to detect genome rearrangements have evolved from microscopic examinations of chromosomes to the more recent microarray-based approaches  The availability of next-generation sequencing technologies may provide a means for scrutinizing entire cancer genomes and transcriptomes at unparalleled resolution  Here we review the methods that have been used to detect genome rearrangements and discuss the scope and limitations of each approach  We end with a discussion of the potential that next-generation sequencing technologies may offer to the field 
                Restriction digest fingerprinting is a common method for characterizing large insert genomic clones  e g   bacterial artificial chromosome (BAC)  P1 artificial chromosome (PAC) and Fosmid clones  This clone fingerprinting method has been widely applied in the construction of clone-based physical maps  which have been used as positional cloning resources as well as to support directed and genome-wide sequencing efforts  This unit describes a robust  large-scale procedure for generation of agarose gel-based clone fingerprints from BAC clones 
                MicroRNAs (miRNAs) are emerging as important  albeit poorly characterized  regulators of biological processes  Key to further elucidation of their roles is the generation of more complete lists of their numbers and expression changes in different cell states  Here  we report a new method for surveying the expression of small RNAs  including microRNAs  using Illumina sequencing technology  We also present a set of methods for annotating sequences deriving from known miRNAs  identifying variability in mature miRNA sequences  and identifying sequences belonging to previously unidentified miRNA genes  Application of this approach to RNA from human embryonic stem cells obtained before and after their differentiation into embryoid bodies revealed the sequences and expression levels of 334 known plus 104 novel miRNA genes  One hundred seventy-one known and 23 novel microRNA sequences exhibited significant expression differences between these two developmental states  Owing to the increased number of sequence reads  these libraries represent the deepest miRNA sampling to date  spanning nearly six orders of magnitude of expression  The predicted targets of those miRNAs enriched in either sample shared common features  Included among the high-ranked predicted gene targets are those implicated in differentiation  cell cycle control  programmed cell death  and transcriptional regulation 
                To facilitate discovery of novel human embryonic stem cell (ESC) transcripts  we generated 2 5 million LongSAGE tags from 9 human ESC lines  Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells  and identified novel ESC transcripts  at least one of which may represent a marker of the pluripotent state 
                We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of &gt 298 high-quality SAGE libraries and &gt 30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection  Several RNA isolation methods were used to handle the diversity of samples processed  and various measures were applied to minimize ditag PCR carryover contamination  Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies  Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure  linker or cross-species ditag contamination  and yield of high-quality tags per sequence read  Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy  From the libraries generated  we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags  Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts  Rather than the usual practice of discarding such tags  we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data  which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling 
                The cause of mental retardation in one-third to one-half of all affected individuals is unknown  Microscopically detectable chromosomal abnormalities are the most frequently recognized cause  but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause  Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants  We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis  by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays  We found de novo deletions as small as 178 kb in eight cases  de novo duplications as small as 1 1 Mb in two cases  and unsuspected mosaic trisomy 9 in another case  This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation 
                We present the results of a simple  statistical assay that measures the G+C content sensitivity bias of gene expression experiments without the requirement of a duplicate experiment  We analyse five gene expression profiling methods: Affymetrix GeneChip  Long Serial Analysis of Gene Expression (LongSAGE)  LongSAGELite  'Classic' Massively Parallel Signature Sequencing (MPSS) and 'Signature' MPSS  We demonstrate the methods have systematic and random errors leading to a different G+C content sensitivity  The relationship between this experimental error and the G+C content of the probe set or tag that identifies each gene influences whether the gene is detected and  if detected  the level of gene expression measured  LongSAGE has the least bias  while Signature MPSS shows a strong bias to G+C rich tags and Affymetrix data show different bias depending on the data processing method (MAS 5 0  RMA or GC-RMA)  The bias in the Affymetrix data primarily impacts genes expressed at lower levels  Despite the larger sampling of the MPSS library  SAGE identifies significantly more genes (60% more RefSeq genes in a single comparison) 
                We analyzed 8 55 million LongSAGE tags generated from 72 libraries  Each LongSAGE library was prepared from a different mouse tissue  Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of approximately 24 000 previously undescribed genomic loci  The visual cortex  pancreas  mammary gland  preimplantation embryo  and placenta contain the largest number of differentially expressed transcripts  25% of which are previously undescribed loci 
                The Mammalian Gene Collection (MGC) consortium (http://mgc nci nih gov) seeks to establish publicly available collections of full-ORF cDNAs for several organisms of significance to biomedical research  including human  To date over 15 200 human cDNA clones containing full-length open reading frames (ORFs) have been identified via systematic expressed sequence tag (EST) analysis of a diverse set of cDNA libraries  however  further systematic EST analysis is no longer an efficient method for identifying new cDNAs  As part of our involvement in the MGC program  we have developed a scalable method for targeted recovery of cDNA clones to facilitate recovery of genes absent from the MGC collection  First  cDNA is synthesized from various RNAs  followed by polymerase chain reaction (PCR) amplification of transcripts in 96-well plates using gene-specific primer pairs flanking the ORFs  Amplicons are cloned into a sequencing vector  and full-length sequences are obtained  Sequences are processed and assembled using Phred and Phrap  and analyzed using Consed and a number of bioinformatics methods we have developed  Sequences are compared with the Reference Sequence (RefSeq) database  and validation of sequence discrepancies is attempted using other sequence databases including dbEST and dbSNP  Clones with identical sequence to RefSeq or containing only validated changes will become part of the MGC human gene collection  Clones containing novel splice variants or polymorphisms have also been identified  Our approach to clone recovery  applied at large scale  has the potential to recover many and possibly most of the genes absent from the MGC collection 
                Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map  genome sequence assembly and BAC end sequences  we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome  The clone set provides coverage for at least 98% of the human fingerprint map  99% of the current assembled sequence and has an effective resolving power of 79 kb  We have made the clone set publicly available  anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease 
                Fingerprinted clone physical maps have proven useful in various applications  supporting both whole-genome and region-specific DNA sequencing as well as gene cloning studies  Fingerprint maps have been generated for several genomes  including those of human  mouse  rat  the nematodes Caenorhabditis elegans and Caenorhabditis briggsae  Arabidopsis thaliana and rice  Fingerprint maps of other genomes  including those of fungi  bacteria  poplar  and the cow  are being generated  The increasing use of fingerprint maps in genomic research has spawned a need in the research community for intuitive computer tools that facilitate viewing of the maps and the underlying fingerprint data  In this report we describe a new Java-based application called iCE (Internet Contig Explorer) that has been designed to provide views of fingerprint maps and associated data  Users can search for and display individual clones  contigs  clone fingerprints  clone insert sizes and markers  Users can also load into the software lists of particular clones of interest and view their fingerprints  iCE is being used at our Genome Centre to offer up to the research community views of the mouse  rat  bovine  C  briggsae  and several fungal genome bacterial artificial chromosome (BAC) fingerprint maps we have either completed or are currently constructing  We are also using iCE as part of the Rat Genome Sequencing Project to manage our provision of rat BAC clones for sequencing at the Human Genome Sequencing Center at the Baylor College of Medicine 
                Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels  We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size  with model parameters determined empirically from &quot marker&quot  lanes containing molecular size standards  Automated identification of restriction fragments involves several steps  including: image preprocessing  to put the data in a form consistent with a linear model  marker lane analysis  for determination of the model parameters  and data lane analysis  a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility  In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones  sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30 000 bp  The integrated suite of software tools  written in MATLAB and collectively called BandLeader  is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center  and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute  Employed in a production mode at the GSC  BandLeader has been used to perform automated restriction fragment identification for more than 850 000 BAC clones for mouse  rat  bovine  and poplar fingerprint mapping projects 
                Programmed cell death (PCD)  important in normal animal physiology and disease  can be divided into at least two morphological subtypes  including type I  or apoptosis  and type II  or autophagic cell death  While many molecules involved in apoptosis have been discovered and studied intensively during the past decade  autophagic cell death is not well characterized molecularly  Here we report the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo  During Drosophila metamorphosis  the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade  To identify and analyze the genes expressed  we examined wild-type patterns of gene expression in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE) [7]  1244 transcripts  including genes involved in autophagy  defense response  cytoskeleton remodeling  noncaspase proteolysis  and apoptosis  were expressed differentially prior to salivary gland death  Mutant expression analysis indicated that several of these genes were regulated by E93  a gene required for salivary gland cell death  Our analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis  and that there are important differences 
                The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene  ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones  which then were sequenced to high accuracy  The MGC has currently sequenced and verified the full ORF for a nonredundant set of &gt 9 000 human and &gt 6 000 mouse genes  Candidate full-ORF clones for an additional 7 800 human and 3 500 mouse genes also have been identified  All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc nci nih gov) 
                Gene expression in a developmentally arrested  long-lived dauer population of Caenorhabditis elegans was compared with a nondauer (mixed-stage) population by using serial analysis of gene expression (SAGE)  Dauer (152 314) and nondauer (148 324) SAGE tags identified 11 130 of the predicted 19 100 C  elegans genes  Genes implicated previously in longevity were expressed abundantly in the dauer library  and new genes potentially important in dauer biology were discovered  Two thousand six hundred eighteen genes were detected only in the nondauer population  whereas 2016 genes were detected only in the dauer  showing that dauer larvae show a surprisingly complex gene expression profile  Evidence for differentially expressed gene transcript isoforms was obtained for 162 genes  H1 histones were differentially expressed  raising the possibility of alternative chromatin packaging  The most abundant tag from dauer larvae (20-fold more abundant than in the nondauer profile) corresponds to a new  unpredicted gene we have named tts-1 (transcribed telomere-like sequence)  which may interact with telomeres or telomere-associated proteins  Abundant antisense mitochondrial transcripts (2% of all tags)  suggest the existence of an antisense-mediated regulatory mechanism in C  elegans mitochondria  In addition to providing a robust tool for gene expression studies  the SAGE approach already has provided the advantage of new gene/transcript discovery in a metazoan