A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR

Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.

PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Background Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. Methods We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Key Results Assembly of the 221 746 sequence reads resulted in 12 442 contigs. Over 50 % (6183) of 12 442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. Conclusions This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines

The CDKN2 gene, encoding the cyclin dependent kinase inhibitor p16, is a tumour suppressor gene involved in melanoma and maps to chromosome band 9p22. Mutations or interstitial deletions of this gene have been found both in the germline of familial melanoma cases and somatically in melanoma cell lines. Previous mutation analyses of melanoma cell lines have indicated a high frequency of C:G to T:A transitions, with all of these mutations occurring at dipyrimidine sites. Including three melanoma cell lines carrying tandem CC to TT mutations, the spectrum of mutations so far reported indicates a possible role for u.v. radiation in the mutagenesis of this gene in some tumours. To further examine this hypothesis we have characterised mutations of the CDKN2 gene in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous deletions of the gene. Of the remaining cell lines, eight were shown by direct sequencing of PCR products from exon 1 and exon 2 to carry a total of nine different mutations of CDKN2. Two cell lines carried tandem CC to TT mutations and a high rate of C:G to T:A transitions was observed. This study provides further evidence for the role of u.v. light in the genesis of melanoma, with one target being the CDKN2 tumour suppressor gene.

Cloning and characterization of a novel human gene related to vascular endothelial growth factor

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells

Purpose: Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse. Experimental Design: We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin γ2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse. Results: Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors. Conclusion: The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer. © 2006 American Association for Cancer Research.

Beyond barcoding : a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus)

The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections.

miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data

miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This article describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/research/software/mirdeep-star

Supplementary Information : Hogan, Holland, Holloway, Petit and Read : Read Classification for Next Generation Sequencing, ESANN 2013, April 2013

This item provides supplementary materials for the paper mentioned in the title, specifically a range of organisms used in the study. The full abstract for the main paper is as follows: Next Generation Sequencing (NGS) technologies have revolutionised molecular biology, allowing clinical sequencing to become a matter of routine. NGS data sets consist of short sequence reads obtained from the machine, given context and meaning through downstream assembly and annotation. For these techniques to operate successfully, the collected reads must be consistent with the assumed species or species group, and not corrupted in some way. The common bacterium Staphylococcus aureus may cause severe and life-threatening infections in humans,with some strains exhibiting antibiotic resistance. In this paper, we apply an SVM classifier to the important problem of distinguishing S. aureus sequencing projects from alternative pathogens, including closely related Staphylococci. Using a sequence k-mer representation, we achieve precision and recall above 95%, implicating features with important functional associations.