Patrick J. Biggs

A conditional knockout resource for the genome-wide study of mouse gene function

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data

BackgroundIlluminas second-generation sequencing platform is playing an increasingly prominent role in modern DNA and RNA sequencing efforts. However, rapid, simple, standardized and independent measures of run quality are currently lacking, as are tools to process sequences for use in downstream applications based on read-level quality data.ResultsWe present SolexaQA, a user-friendly software package designed to generate detailed statistics and at-a-glance graphics of sequence data quality both quickly and in an automated fashion. This package contains associated software to trim sequences dynamically using the quality scores of bases within individual reads.ConclusionThe SolexaQA package produces standardized outputs within minutes, thus facilitating ready comparison between flow cell lanes and machine runs, as well as providing immediate diagnostic information to guide the manipulation of sequence data for downstream analyses.

Identification of the familial cylindromatosis tumour-suppressor gene

Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein–glycine-conserved (CAP–GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).

Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours

Testicular germ-cell tumours (TGCT) affect 1 in 500 men and are the most common cancer in males aged 15–40 in Western European populations. The incidence of TGCT has risen dramatically over the last century. Known risk factors for TGCT include a history of undescended testis (UDT), testicular dysgenesis, infertility, previously diagnosed TGCT (ref. 7) and a family history of the disease. Brothers of men with TGCT have an 8-10-fold risk of developing TGCT (refs 8,9), whereas the relative risk to fathers and sons is fourfold (ref. 9). This familial relative risk is much higher than that for most other types of cancer. We have collected samples from 134 families with two or more cases of TGCT, 87 of which are affected sibpairs. A genome-wide linkage search yielded a heterogeneity lod (hlod) score of 2.01 on chromosome Xq27 using all families compatible with X inheritance. We obtained a hlod score of 4.7 from families with at least one bilateral case, corresponding to a genome-wide significance level of P=0.034. The proportion of families with UDT linked to this locus was 73% compared with 26% of families without UDT (P=0.03). Our results provide evidence for a TGCT susceptibility gene on chromosome Xq27 that may also predispose to UDT.

Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer.

Thyroid goiter is a common condition that is often associated with iodine deficiency. Familial forms of goiter in areas not known to feature iodine deficiency are much less common. We have performed a genomic search on a single large Canadian family with 18 cases of nontoxic multinodular goiter in which 2 individuals also had papillary lesions highly suggestive of papillary carcinoma. A locus on chromosome 14q (MNG1 [multinodular goiter 1]) has been identified, with a maximal two-point LOD score of 3.8 at D14S1030 and a multipoint LOD score of 4.88 at the same marker, defined by D14S1062 (upper boundary) and D14S267 (lower boundary). The gene encoding thyroid-stimulating hormone receptor (TSHR), which is located on chromosome 14q, is outside the linked region. To determine the role of this gene in familial nonmedullary thyroid cancer (NMTC), we studied 37 smaller pedigrees each containing at least two cases of NMTC. Analysis by both parametric and nonparametric methods indicates that only a very small proportion of familial NMTC (point estimate 0.001, support intervals 0-.6 under a dominant model) is attributable to MNG1.

Mutagenic insertion and chromosome engineering resource (MICER).

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.

Systematic Error in Seed Plant Phylogenomics

Resolving the closest relatives of Gnetales has been an enigmatic problem in seed plant phylogeny. The problem is known to be difficult because of the extent of divergence between this diverse group of gymnosperms and their closest phylogenetic relatives. Here, we investigate the evolutionary properties of conifer chloroplast DNA sequences. To improve taxon sampling of Cupressophyta (non-Pinaceae conifers), we report sequences from three new chloroplast (cp) genomes of Southern Hemisphere conifers. We have applied a site pattern sorting criterion to study compositional heterogeneity, heterotachy, and the fit of conifer chloroplast genome sequences to a general time reversible + G substitution model. We show that non-time reversible properties of aligned sequence positions in the chloroplast genomes of Gnetales mislead phylogenetic reconstruction of these seed plants. When 2,250 of the most varied sites in our concatenated alignment are excluded, phylogenetic analyses favor a close evolutionary relationship between the Gnetales and Pinaceae—the Gnepine hypothesis. Our analytical protocol provides a useful approach for evaluating the robustness of phylogenomic inferences. Our findings highlight the importance of goodness of fit between substitution model and data for understanding seed plant phylogeny.

The Evolutionary Root of Flowering Plants

Correct rooting of the angiosperm radiation is both challenging and necessary for understanding the origins and evolution of physiological and phenotypic traits in flowering plants. The problem is known to be difficult due to the large genetic distance separating flowering plants from other seed plants and the sparse taxon sampling among basal angiosperms. Here, we provide further evidence for concern over substitution model misspecification in analyses of chloroplast DNA sequences. We show that support for Amborella as the sole representative of the most basal angiosperm lineage is founded on sequence site patterns poorly described by time-reversible substitution models. Improving the fit between sequence data and substitution model identifies Trithuria, Nymphaeaceae, and Amborella as surviving relatives of the most basal lineage of flowering plants. This finding indicates that aquatic and herbaceous species dominate the earliest extant lineage of flowering plants. [; ; ; ; ; .].

AN APPROACH TO TRANSCRIPTOME ANALYSIS OF NON-MODEL ORGANISMS USING SHORT-READ SEQUENCES

Transcriptome analysis using high-throughput short-read sequencing technology is straightforward when the sequenced genome is the same species or extremely similar to the reference genome. We present an analysis approach for when the sequenced organism does not have an already sequenced genome that can be used for a reference, as will be the case of many non-model organisms. As proof of concept, data from Solexa sequencing of the polyploid plant Pachycladon enysii was analysed using our approach with its nearest model reference genome being the diploid plant Arabidopsis thaliana. By using a combination of mapping and de novo assembly tools we could determine duplicate genes belonging to one or other of the genome copies. Our approach demonstrates that transcriptome analysis using high-throughput short-read sequencing need not be restricted to the genomes of model organisms.

High Throughput Genome-Wide Survey of Small RNAs from the Parasitic Protists Giardia intestinalis and Trichomonas vaginalis

RNA interference (RNAi) is a set of mechanisms which regulate gene expression in eukaryotes. Key elements of RNAi are small sense and antisense RNAs from 19 to 26 nt generated from double-stranded RNAs. MicroRNAs (miRNAs) are a major type of RNAi-associated small RNAs and are found in most eukaryotes studied to date. To investigate whether small RNAs associated with RNAi appear to be present in all eukaryotic lineages, and therefore present in the ancestral eukaryote, we studied two deep-branching protozoan parasites, Giardia intestinalis and Trichomonas vaginalis. Little is known about endogenous small RNAs involved in RNAi of these organisms. Using Illumina Solexa sequencing and genome-wide analysis of small RNAs from these distantly related deep-branching eukaryotes, we identified 10 strong miRNA candidates from Giardia and 11 from Trichomonas. We also found evidence of Giardia short-interfering RNAs potentially involved in the expression of variant-specific surface proteins. In addition, eight new small nucleolar RNAs from Trichomonas are identified. Our results indicate that miRNAs are likely to be general in ancestral eukaryotes and therefore are likely to be a universal feature of eukaryotes.