Identification of piggyBac-mediated insertions in Plasmodium berghei by next generation sequencing.
ABSTRACT: BACKGROUND: The piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening. The identification of genotype of piggyBac mutants in the Plasmodium genome is thus an indispensable step in forward genetic analysis. Several PCR-based approaches have been used to identify the piggyBac insertion sites in Plasmodium falciparum and Plasmodium berghei, but all are tedious and inefficient. Next generation sequencing can produce large amounts of sequence data and is particularly suitable for genome-wide association studies. In this study, the Next generation sequencing technology was employed to efficiently identify piggyBac insertion sites in the genome of P. berghei. METHODS: Plasmodium berghei parasites were co-transfected with piggyBac donor and helper plasmids. Initially, the classical inverse PCR method was used to identify the existence of piggyBac insertions in the P. berghei genome. The whole genome of post-transfection parasites was subsequently sequenced with a PCR-free paired-end module using the Illumina HiSeq sequencing system. The two distinct methods ('BLAST method' and 'SOAP method') were employed to identify piggyBac insertion sites in the P. berghei genome with Illumina sequencing data. All the identified piggyBac insertions were further tested by half-nested PCR. RESULTS: The inverse PCR method resulted in a very low yield of ten individual insertions identified. Conversely, 47 piggyBac insertions were identified from about 1 Gb of Illumina sequencing data via the two distinct analysis methods. The majority of identified piggyBac insertions were confirmed by half-nested PCR. In addition, 1,850 single nucleotide polymorphisms were identified through alignment of the Illumina sequencing data of the P. berghei ANKA strain used in this study with the reference genome sequences. CONCLUSION: This study demonstrates that a high-throughput genome sequencing approach is an efficient tool for the identification of piggyBac-mediated insertions in Plasmodium parasites.
Project description:The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo.We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated.These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo.
Project description:The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.
Project description:<h4>Background</h4>Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.<h4>Results</h4>In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.<h4>Conclusion</h4>Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.
Project description:The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.
Project description:Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED 90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED 90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca 2+/H + antiporter. Conclusions: Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.
Project description: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 >10? reads corresponding to >10? 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.
Project description:BACKGROUND:Transposable elements (TEs) are major components of eukaryotic genomes and drivers of genome evolution, producing intraspecific polymorphism and interspecific differences through mobilization and non-homologous recombination. TE insertion sites are often highly variable within species, creating a need for targeted genome re-sequencing (TGS) methods to identify TE insertion sites. METHODS:We present a hemi-specific PCR approach for TGS of P-elements in Drosophila genomes on the Illumina platform. We also present a computational framework for identifying new insertions from TGS reads. Finally, we describe a new method for estimating the frequency of TE insertions from WGS data, which is based precise insertion sites provided by TGS annotations. RESULTS:By comparing our results to TE annotations based on whole genome re-sequencing (WGS) data for the same Drosophilamelanogaster strain, we demonstrate that TGS is powerful for identifying true insertions, even in repeat-rich heterochromatic regions. We also demonstrate that TGS offers enhanced annotation of precise insertion sites, which facilitates estimation of TE insertion frequency. CONCLUSIONS:TGS by hemi-specific PCR is a powerful approach for identifying TE insertions of particular TE families in species with a high-quality reference genome, at greatly reduced cost as compared to WGS. It may therefore be ideal for population genomic studies of particular TE families. Additionally, TGS and WGS can be used as complementary approaches, with TGS annotations identifying more annotated insertions with greater precision for a target TE family, and WGS data allowing for estimates of TE insertion frequencies, and a broader picture of the location of non-target TEs across the genome.
Project description:Genome sequencing of Anopheles gambiae was completed more than ten years ago and has accelerated research on malaria transmission. However, annotation needs to be refined and verified experimentally, as most predicted transcripts have been identified by comparative analysis with genomes from other species. The mosquito midgut-the first organ to interact with Plasmodium parasites-mounts effective antiplasmodial responses that limit parasite survival and disease transmission. High-throughput Illumina sequencing of the midgut transcriptome was used to identify new genes and transcripts, contributing to the refinement of An. gambiae genome annotation.We sequenced ~223 million reads from An. gambiae midgut cDNA libraries generated from susceptible (G3) and refractory (L35) mosquito strains. Mosquitoes were infected with either Plasmodium berghei or Plasmodium falciparum, and midguts were collected after the first or second Plasmodium infection. In total, 22,889 unique midgut transcript models were generated from both An. gambiae strain sequences combined, and 76% are potentially novel. Of these novel transcripts, 49.5% aligned with annotated genes and appear to be isoforms or pre-mRNAs of reference transcripts, while 50.5% mapped to regions between annotated genes and represent novel intergenic transcripts (NITs). Predicted models were validated for midgut expression using qRT-PCR and microarray analysis, and novel isoforms were confirmed by sequencing predicted intron-exon boundaries. Coding potential analysis revealed that 43% of total midgut transcripts appear to be long non-coding RNA (lncRNA), and functional annotation of NITs showed that 68% had no homology to current databases from other species. Reads were also analyzed using de novo assembly and predicted transcripts compared with genome mapping-based models. Finally, variant analysis of G3 and L35 midgut transcripts detected 160,742 variants with respect to the An. gambiae PEST genome, and 74% were new variants. Intergenic transcripts had a higher frequency of variation compared with non-intergenic transcripts.This in-depth Illumina sequencing and assembly of the An. gambiae midgut transcriptome doubled the number of known transcripts and tripled the number of variants known in this mosquito species. It also revealed existence of a large number of lncRNA and opens new possibilities for investigating the biological function of many newly discovered transcripts.
Project description:Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.
Project description:In this study, we investigated the mutation tendency of a mutator rodent malaria parasite, Plasmodium berghei, with proofreading-deficient DNA polymerase ?. Wild-type and mutator parasites were maintained in mice for over 24 weeks, and the genome-wide accumulated mutations were determined by high-throughput sequencing. The mutator P. berghei had a significant preference for C/G to A/T substitutions; thus, its genome had a trend towards a higher AT content. The mutation rate was influenced by the sequence context, and mutations were markedly elevated at TCT. Some genes mutated repeatedly in replicate passage lines. In particular, knockout mutations of the AP2-G gene were frequent, which conferred strong growth advantages on parasites during the blood stage but at the cost of losing the ability to form gametocytes. This is the first report to demonstrate a biased mutation tendency in malaria parasites, and its results help to promote our basic understanding of Plasmodium genetics.