Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

Improving the Efficiency of Transposon Mutagenesis in Salmonella Enteritidis by Overcoming Host-Restriction Barriers

Transposon mutagenesis using transposome complex is a powerful method for functional genomics analysis in diverse bacteria by creating a large number of random mutants to prepare a genome-saturating mutant library. However, strong host restriction barriers can lead to limitations with species- or strain-specific restriction-modification systems. The purpose of this study was to enhance the transposon mutagenesis efficiency of Salmonella Enteritidis to generate a larger number of random insertion mutants. Host-adapted Tn5 DNA was used to form a transposome complex, and this simple approach significantly and consistently improved the efficiency of transposon mutagenesis, resulting in a 46-fold increase in the efficiency as compared to non-adapted transposon DNA fragments. Random nature of Tn5 insertions was confirmed by high-throughput sequencing of the Tn5-junction sequences. The result based on S. Enteritidis in this study should find broad applications in preparing a comprehensive mutant library of other species using transposome complex.     

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.     

The LORE1 insertion mutant resource

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) ( http://lotus.au.dk ). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG‐hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost‐efficient strategy for generation of non‐transgenic mutant collections for unrestricted use in plant research.     

Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En-I transposon system

Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer-Inhibitor (En-I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny.     

Use of the transposon Ac as a gene-searching engine in the maize genome

We show here that, although genes constitute only a small percentage of the maize genome, it is possible to identify them phenotypically as Ac receptor sites. Simple and efficient Ac transposition assays based on the well-studied endosperm markers bz and wx were used to generate a collection of >1300 independent Ac transposants. The majority of transposed Ac elements are linked to either the bz or the wx donor loci on chromosome 9. A few of the insertions produce obvious visible phenotypes, but most of them do not, suggesting that these populations will be more useful for reverse genetics than for forward transposon mutagenesis. An inverse polymerase chain reaction method was adapted for the isolation of DNA adjacent to the transposed Ac elements (tac sites). Most Ac insertions were into unique DNA. By sequencing tac sites and comparing the sequences to existing databases, insertions were identified in a number of putative maize genes. The expression of most of these genes was confirmed by RNA gel blot analysis. We report here the isolation and characterization of the first 46 tac sites from the two insertion libraries.     

ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data

High-throughput analysis of genome-wide random transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next-generation sequencing approaches, e.g. Tn-seq/INseq, HITS and TraDIS, have been developed that accurately map the site of transposon insertions by mutant-specific amplification and sequence readout of DNA flanking the transposon insertions site, assigning a measure of essentiality based on the number of reads per insertion site flanking sequence or per gene. However, analysis of these large and complex datasets is hampered by the lack of an easy to use and automated tool for transposon insertion sequencing data. To fill this gap, we developed ESSENTIALS, an open source, web-based software tool for researchers in the genomics field utilizing transposon insertion sequencing analysis. It accurately predicts (conditionally) essential genes and offers the flexibility of using different sample normalization methods, genomic location bias correction, data preprocessing steps, appropriate statistical tests and various visualizations to examine the results, while requiring only a minimum of input and hands-on work from the researcher. We successfully applied ESSENTIALS to in-house and published Tn-seq, TraDIS and HITS datasets and we show that the various pre- and post-processing steps on the sequence reads and count data with ESSENTIALS considerably improve the sensitivity and specificity of predicted gene essentiality.     

TILLING在水稻育种中的应用前景

TILLING(Targeting Induced Local Lesions in Genomes)是功能基因组研究中应用的一种反向遗传学技术。它能高通量低成本地在EMS诱变群体中鉴定出发生在特定基因上的点突变。在其基础上发展出的EcoTILLING技术则可发现种质资源中的SNP位点及小插入或缺失多态性位点。水稻是非常重要的粮食作物, 也是已经完成了全基因组序列测定,有丰富的生物信息学资源可以利用的基因组研究模式植物。水稻的分子标记辅助育种将在育种中扮演越来越重要的角色。在这样的背景下,本文从基于特定基因的种质资源鉴定、EMS诱变育种、及水稻功能标记开发等方面论述了其在水稻育种中的应用前景。     

Steady-state transposon mutagenesis in inbred maize

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.     

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

SNP analysis to dissect human traits

The analysis of complex human diseases has been spurred by the number of published genomic sequence variants - many identified in the course of sequencing the human genome. But, to be useful for genetic analysis, variants have to be mapped accurately, their frequencies in various populations determined, and automated high-throughput assay techniques developed. Recently proposed methods address these issues: the use of 'reduced representation shotgun' methods for more efficient detection of single nucleotide polymorphisms (SNPs), the employment of high-throughput genotyping techniques, the development of SNP maps that incorporate information about linkage disequilibrium, and the use of SNPs in identifying susceptibility genes for common illnesses.     

Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin.     

Yet Another Quick Assembly,Analysis and Trimming Tool (YAQAAT): A Server for the Automated Assembly and Analysis of Sanger Sequencing Data

Even with the ubiquity of Sanger sequencing, automated assembly software are predominantly stand-alone software packages for desktop/laptop use with very few online equivalents, thus geospatially constraining sequence analysis and assembly. With increased data output worldwide, there is also a need for automated quality checks and trimming prior to large assemblies, along with automated detection of mutations. Through web servers with expanded automation and functionalities, even smartphones/phablets can be used to perform complex analysis previously limited to desktops, especially if they can upload files from cloud storage. To facilitate such online accessible sequence assembly and analysis, we created Yet Another Quick Assembly, Analysis and Trimming Tool web server for the automated assembly of multiple .ab1 and .FASTQ sequencing reads de novo with automated trimming and scanning of the assembled sequences for single nucleotide polymorphisms and insertions or deletions without installation of software, allowing it to be accessed from anywhere with Internet access and with minimal dependency on other software and web tools.     

Mos1-mediated insertional mutagenesis in Caenorhabditis elegans

We describe a protocol for mutating genes in the nematode Caenorhabditis elegans using the Mos1 transposon of Drosophila mauritiana. Mutated genes containing a Mos1 insertion are molecularly tagged by this heterologous transposable element. Mos1 insertions can therefore be identified in as little as 3 weeks using only basic molecular biology techniques. Mutagenic efficiency of Mos1 is tenfold lower than classical chemical mutagens. However, the ease and speed with which mutagenic insertions can be mapped compares favorably with the vast amount of work involved in classical genetic mapping. Therefore, Mos1 could be the tool of choice when screening procedures are efficient. In addition, Mos1 mutagenesis can greatly simplify the mapping of mutations that exhibit low penetrance, subtle or synthetic phenotypes. The recent development of targeted engineering of C. elegans loci carrying Mos1 insertions further increases the attractiveness of Mos1-mediated mutagenesis.     

Genome‐wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato

Genome‐wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro‐Tom genome were identified by a whole‐genome shotgun sequencing analysis to estimate the spectrum and distribution of whole‐genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired‐end reads for four EMS‐induced mutants and three gamma‐ray‐irradiated lines as well as a wild‐type line were obtained by next‐generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma‐ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1 140 687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild‐type Micro‐Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild‐type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse‐genetic approaches.     

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.     

Construction of a single-transposon-insertion mutant in Rhizobium sp. strain TAL1145 from a double-insertion mutant

A method for developing a single-transposon-insertion mutant from a double-insertion mutant in Rhizobium is described. An exopolysaccharide (EPS)-defective mutant containing two Tn 5-lacZ insertions was complemented with cloned wild-type DNA for EPS synthesis. One of the Tn 5-lacZ insertions from the mutant was transferred to the complementing plasmid by homologous recombination. The plasmid containing the Tn 5-lacZ insertion in the gene involved in EPS synthesis was transferred into the wild-type strain and the Tn 5-lacZ was homogenized to obtain an EPS-defective mutant with a single Tn 5-lacZ insertion.