Sequence Traces Research Articles

Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus

The mimetic wing patterns of Heliconius butterflies are an excellent example of both adaptive radiation and convergent evolution. Alleles at the HmYb and HmSb loci control the presence/absence of hindwing bar and hindwing margin phenotypes respectively between divergent races of Heliconius melpomene, and also between sister species. Here, we used fine-scale linkage mapping to identify and sequence a BAC tilepath across the HmYb/Sb loci. We also generated transcriptome sequence data for two wing pattern forms of H. melpomene that differed in HmYb/Sb alleles using 454 sequencing technology. Custom scripts were used to process the sequence traces and generate transcriptome assemblies. Genomic sequence for the HmYb/Sb candidate region was annotated both using the MAKER pipeline and manually using transcriptome sequence reads. In total, 28 genes were identified in the HmYb/Sb candidate region, six of which have alternative splice forms. None of these are orthologues of genes previously identified as being expressed in butterfly wing pattern development, implying previously undescribed molecular mechanisms of pattern determination on Heliconius wings. The use of next-generation sequencing has therefore facilitated DNA annotation of a poorly characterized genome, and generated hypotheses regarding the identity of wing pattern at the HmYb/Sb loci.

OLFinder—a program which disentangles DNA sequences containing heterozygous indels

The presence of heterozygous indels in a DNA sequence usually results in the sequence being discarded. If the sequence trace is of high enough quality, however, it will contain enough information to reconstruct the two constituent sequences with very little ambiguity. Solutions already exist using comparisons with a known reference sequence, but this is often unavailable for nonmodel organisms or novel DNA regions. I present a program which determines the sizes and positions of heterozygous indels in a DNA sequence and reconstructs the two constituent haploid sequences. No external data such as a reference sequence or other prior knowledge are required. Simulation suggests an accuracy of >99% from a single read, with errors being eliminable by the inclusion of a second sequencing read, such as one using a reverse primer. Diploid sequences can be fully reconstructed across any number of heterozygous indels, with two overlapping sequencing reads almost always sufficient to infer the entire DNA sequence. This eliminates the need for costly and laborious cloning, and allows data to be used which would otherwise be discarded. With no more laboratory work than is needed to produce two normal sequencing reads, two aligned haploid sequences can be produced quickly and accurately and with extensive phasing information.

Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Various technologies are currently available to quantify DNA methylation. However, rapid and simple methods for determining the DNA methylation status of CpG sites in genes still remain elusive. In this report, we describe a novel method for the rapid quantification of CpG methylation on the basis of direct bisulfite-PCR sequencing method. According to the principles of bisulfite-PCR, converting unmethylated cytosines to thymine while leaving methylated cytosines unchanged, we regard the CpG site as a SNP and estimate the methylation status of cytosines in the given CG dinucleotides by measuring the ratio of the cytosine peak height to the sum of cytosine and thymine peak heights in automated DNA sequencing traces. Furthermore, we take several effective measures to break through the ‘bottleneck' problems that render the routine bisulfite sequencing method unsuitable for quantitative methylation. In comparison with pyrosequencing and bisulfite-cloning sequencing, our method is confirmed to be a simple, high-throughput and cost-effective technology for determining the methylation status of specific genes. Accordingly, this novel method is anticipated to be an efficient and economical alternative tool for rapid quantification of methylation patterns in screening large numbers of clinical samples across multiple genes.

Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler.

BackgroundThe rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage.ResultsWe have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli.ConclusionThe statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

MouseIndelDB: a database integrating genomic indel polymorphisms that distinguish mouse strains

MouseIndelDB is an integrated database resource containing thousands of previously unreported mouse genomic indel (insertion and deletion) polymorphisms ranging from ∼100 nt to 10 Kb in size. The database currently includes polymorphisms identified from our alignment of 26 million whole-genome shotgun sequence traces from four laboratory mouse strains mapped against the reference C57BL/6J genome using GMAP. They can be queried on a local level by chromosomal coordinates, nearby gene names or other genomic feature identifiers, or in bulk format using categories including mouse strain(s), class of polymorphism(s) and chromosome number. The results of such queries are presented either as a custom track on the UCSC mouse genome browser or in tabular format. We anticipate that the MouseIndelDB database will be widely useful for research in mammalian genetics, genomics, and evolutionary biology. Access to the MouseIndelDB database is freely available at: http://variation.osu.edu/.

Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus : Implications for Molecular Diagnosis in Clinical Microbiology Laboratories

Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping.

Improvements to services at the European Nucleotide Archive

The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe’s primary nucleotide sequence archival resource, safeguarding open nucleotide data access, engaging in worldwide collaborative data exchange and integrating with the scientific publication process. ENA has made significant contributions to the collaborative nucleotide archival arena as an active proponent of extending the traditional collaboration to cover capillary and next-generation sequencing information. We have continued to co-develop data and metadata representation formats with our collaborators for both data exchange and public data dissemination. In addition to the DDBJ/EMBL/GenBank feature table format, we share metadata formats for capillary and next-generation sequencing traces and are using and contributing to the NCBI SRA Toolkit for the long-term storage of the next-generation sequence traces. During the course of 2009, ENA has significantly improved sequence submission, search and access functionalities provided at EMBL–EBI. In this article, we briefly describe the content and scope of our archive and introduce major improvements to our services.

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

Intraocular penetration of voriconazole and caspofungin in a patient with fungal endophthalmitis

and L180M are compensatory mutations that increase the fitness of hepatitis B viruses harbouring mutations in the YMDD domain of the viral polymerase. It is of interest that while the YMDD domain mutation M204V was present as a minority species at baseline, V173L and L180M co-existed with the wild type as a mixed sequence in these two positions. While it is known that entecavir does not select lamivudine-associated mutations de novo, it is clear from this patient that pre-existing mutations were selected rapidly and resulted in virological rebound. Failure of entecavir was said to be rare and to occur only after 1–2 years of therapy. Hence, this is a case of early virological hepatitis B rebound on entecavir, through selection of lamivudine-associated mutations, but without developing any further entecavir-associated mutations in a treatment-naive patient. We feel it is essential to perform baseline resistance profiling on all patients started on entecavir even if treatment naive. The sequence tracing also needs to be carefully scrutinized as the mutations may appear as a minority species not obviously visible on population sequencing analysis.

Identification of 1,531 cSNPs from Full-length Enriched cDNA Libraries of the Korean Native Pig Using in Silico Analysis

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomics related studies, genome annotation and SNP discovery. We analyzed 7,392 high-quality chromatograms (Phred value ≥30) obtained from sequencing the 5' ends of clones derived from full-length enriched cDNA libraries of Korean native pigs including brainstem, liver, cerebellum, neocortex and spleen libraries. In addition, 50,000 EST sequence trace files obtained from GenBank were combined with our sequences to identify cSNPs in silico. The process generated 11,324 contigs, of which 2,895 contigs contained at least one SNP and among them 610 contigs had a minimum of one sequence from Korean native pigs. Of 610 contigs, we randomly selected 262 contigs and performed in silico analysis for the identifica tion of cSNPs. From the results, we identified 1,531 putative coding single nucleotide polymorphisms (cSNPs) and the SNP detection frequency was one SNP per 465 bp. A large-scale sequencing result of clones from full-length enriched cDNA libraries and identified cSNPs will serve as a useful resource to functional genomics related projects such as a pig HapMap project in the near future.

Meningococcus genome informatics platform: a system for analyzing multilocus sequence typing data

The Meningococcus Genome Informatics Platform (MGIP) is a suite of computational tools for the analysis of multilocus sequence typing (MLST) data, at http://mgip.biology.gatech.edu. MLST is used to generate allelic profiles to characterize strains of Neisseria meningitidis, a major cause of bacterial meningitis worldwide. Neisseria meningitidis strains are characterized with MLST as specific sequence types (ST) and clonal complexes (CC) based on the DNA sequences at defined loci. These data are vital to molecular epidemiology studies of N. meningitidis, including outbreak investigations and population biology. MGIP analyzes DNA sequence trace files, returns individual allele calls and characterizes the STs and CCs. MGIP represents a substantial advance over existing software in several respects: (i) ease of use—MGIP is user friendly, intuitive and thoroughly documented; (ii) flexibility—because MGIP is a website, it is compatible with any computer with an internet connection, can be used from any geographic location, and there is no installation; (iii) speed—MGIP takes just over one minute to process a set of 96 trace files; and (iv) expandability—MGIP has the potential to expand to more loci than those used in MLST and even to other bacterial species.

Evolution of Phage-Type RNA Polymerases in Higher Plants: Characterization of the Single Phage-Type RNA Polymerase Gene from Selaginella moellendorffii

Selaginella moellendorfii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases (RpoTs) were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3006 nt, encoding a putative protein of 1002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. In phylogenetic analyses, the Selaginella RpoT polymerase is in a sister position to all other phage-type polymerases of angiosperms. However, according to its conserved exon-intron structure, the Selaginella RpoT gene is representative of the molecular evolutionary lineage giving rise to the RpoT gene family of flowering plants. The N-terminal transit peptide of SmRpoT is shown to confer targeting of green fluorescent protein exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. Angiosperms and the moss P. patens possess small gene families encoding RpoTs, which include mitochondrial- and chloroplast-targeted RNA polymerases. In striking contrast, the Selaginella RpoT gene is shown to be single-copy, although Selaginella, as a lycophyte, has a phylogenetic position between Physcomitrella and angiosperms. Thus, there is no evidence that Selaginella may contain a nuclear-encoded phage-type chloroplast RNA polymerase.

A Domestic cat X Chromosome Linkage Map and the Sex-LinkedorangeLocus: Mapping oforange, Multiple Origins and Epistasis Overnonagouti

A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal of localizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an average spacing of 3 Mb were selected from sequence traces of the cat 1.9x whole genome sequence (WGS), including the pseudoautosomal region 1 (PAR1). Extreme variation in recombination rates (centimorgans per megabase) was observed along the X chromosome, ranging from a virtual absence of recombination events in a region estimated to be >30 Mb to recombination frequencies of 15.7 cM/Mb in a segment estimated to be <0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placing this locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Fine mapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.9x-coverage sequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that could reduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype in the domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.

Reporting 678 putative cSNPs from full‐length enriched cDNA sequences of the Korean native pig

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We have analysed 1970 high-quality chromatograms (Phred value >or= 30) that were obtained from sequencing the 5' ends of brainstem, liver, neocortex and spleen clones derived from full-length enriched cDNA libraries from Korean native pigs. In addition, 50,000 pig expressed sequence tag (EST) sequence trace files were obtained from Genbank and combined with our sequencing information to facilitate SNP identification in silico. The process generated 8118 contigs, of which 239 included minimum one sequence from Korean native pig and contained 678 putative coding single nucleotide polymorphisms (cSNPs). Of these, 33 putative cSNPs were randomly selected for confirmatory analysis and validated using 20 pigs from four different breeds (Duroc, Landrace, Yorkshire, Korean native pig). Of the 33 putative cSNPs, 20 were confirmed (61%), which was similar to the frequency reported in other studies. We also identified 15 new cSNPs from the validation process, which were not detected by our in silico analysis. Our study shows that analysing genetically diverse pig breeds including the Korean native pig could serve as a useful strategy for generating a large number of cSNPs.

Single nucleotide polymorphisms (SNPs) of exons 3, 4, 5 and 9 of the matrix metalloproteinase 2 (MMP-2) gene in breast carcinoma of Malaysian women using high resolution melting analysis

Background Breast cancer metastasis is a factor contributing to high disease mortality. Single nucleotide polymorphism (SNP) of the matrix metalloproteinase 2 (MMP-2) gene, which controls the enzyme for degradation of macromolecules of the connective tissues and extracellular matrix, has been associated with cancer invasion and metastasis. Aim The aim was to detect SNPs of exons 3, 4, 5 and 9 of the MMP-2 gene in breast carcinoma of Malaysian women using high resolution melting analysis (HRM). Methods Genomic DNA was obtained of both tumour mass and normal adjacent tissue from 60 Malaysian breast cancer patients. SNP scanning involving all four exons of MMP-2 coding regions was performed by HRM analysis on Rotor-Gene 6000 real-time PCR. The PCR products presented with aberrant melting curve pattern were purified and sequenced. Sequence trace data obtained were analysed with Staden Package for mutation detection. Results The scanning of the MMP-2 exons detected 4 known SNPs in exon 5 (678G>C, 750C>T), intron 5 (832+12C>T) and exon 9 (1380G>A). No SNP was detected on exon 3 and 4. Five patients carried both copies of variant A SNP 1380G>A while only one patient carried homozygous variant T SNP 832+12C>T. Only one patient with homozygous variant T allele SNP 1380G>A and SNP 678G>C had poor outcome with extensive metastasis and failed chemotherapy. Conclusion A relationship between SNP of MMP-2 with disease progression and poor therapeutic response is a strong possibility. The utilisation of HRM, a sensitive and cost effective method for scanning the MMP-2 gene, is suggested on all exons to elucidate the involvement of SNPs in cancer progression.

DNA Barcoding of Israeli Indigenous and Introduced Cichlids

The objectives of this study were barcoding and taxonomic analysis of the five tilapiine species (Oreochromis aureus, O. niloticus, O. mossam- bicus, Sarotherodon galilaeus, and Tilapia zillii), two tilapia hybrid strains (Florida red tilapia and Philippine red tilapia), and two endemic wild cichlids (Tristramella simonis and Astatotilapia flaviijosephi) available in Israel, as well as O. urolepis hornorum. Cytochrome oxidase subunit I (COI) 619 bp sequence traces of 104 individuals were assembled, aligned, and compared (GenBank project GI 209553463). The DNA sequences of two hybrid strains were identical to those of O. hornorum and O. aureus. Absence of intra-specific variability was detected in the commercially used species, O. aureus, S. galilaeus, O. mossambicus, and O. urolepis horno- rum. Two DNA sequence variants were detected in O. niloticus originating from Ghana and Egypt. In contrast, 2-3 variants were detected in the DNA of each of the non-commercial species. Amino-acid sequences were identical in all “true tilapias” and different from the sequences in the endemic cichlids. As a whole, the protein phylogenetic tree fitted the expected conventional taxonomy as opposed to the respective DNA-based tree. Sequences FJ348047-FJ348150 were submitted to GenBank via the BOLD database (identical to FISH001-08 - FISH104-08 in this database).

DNA barcoding demystified

Only 10% of the earth’s biota has been described despite250 yearsoftaxonomicresearch(Wilson2000).Thisisinlargepart a reflection of the extent and complexity of biologicaldiversity,butitisalsotruethattraditionaltaxonomictechniquesare labourious and highly specialised, and taxonomic expertiseis very thinly spread across the myriad groups of life (Scotland

Unique Features of Nuclear mRNA Poly(A) Signals and Alternative Polyadenylation in Chlamydomonas reinhardtii

To understand nuclear mRNA polyadenylation mechanisms in the model alga Chlamydomonas reinhardtii, we generated a data set of 16,952 in silico-verified poly(A) sites from EST sequencing traces based on Chlamydomonas Genome Assembly v.3.1. Analysis of this data set revealed a unique and complex polyadenylation signal profile that is setting Chlamydomonas apart from other organisms. In contrast to the high-AU content in the 3'-UTRs of other organisms, Chlamydomonas shows a high-guanylate content that transits to high-cytidylate around the poly(A) site. The average length of the 3'-UTR is 595 nucleotides (nt), significantly longer than that of Arabidopsis and rice. The dominant poly(A) signal, UGUAA, was found in 52% of the near-upstream elements, and its occurrence may be positively correlated with higher gene expression levels. The UGUAA signal also exists in Arabidopsis and in some mammalian genes but mainly in the far-upstream elements, suggesting a shift in function. The C-rich region after poly(A) sites with unique signal elements is a characteristic downstream element that is lacking in higher plants. We also found a high level of alternative polyadenylation in the Chlamydomonas genome, with a range of up to 33% of the 4057 genes analyzed having at least two unique poly(A) sites and approximately 1% of these genes having poly(A) sites residing in predicted coding sequences, introns, and 5'-UTRs. These potentially contribute to transcriptome diversity and gene expression regulation.

Expressed Sequence Tags With cDNA Termini: Previously Overlooked Resources for Gene Annotation and Transcriptome Exploration in Chlamydomonas reinhardtii

Many of Chlamydomonas reinhardtii expressed sequence tags (ESTs) in GenBank dbEST and community EST assemblies were either over- or undertrimmed in terms of their cDNA termini, which are defined as the diagnostic sequence elements that delineate 3'/5' ends of mRNA transcripts. Overtrimming represents a loss of directional, positional, and structural information of transcript ends whereas undertrimming causes unclean spurious sequences retained in ESTs that exert deleterious impacts on downstream EST-based applications. We examined 309,278 raw EST sequencing trace files of C. reinhardtii and found that only 57% had cDNA termini that matched the expected structures specified in their cDNA library constructions while satisfying our minimum length requirement for their final clean sequences. Using GMAP, 156,963 individual ESTs were mapped to the genome successfully, with their in silico-verified cDNA termini anchored to the genome. Our data analysis suggested strong macro- and microheterogeneity of 3'/5' end positions of individual transcripts derived from the same genes in C. reinhardtii. This work annotating differential ends of individual transcripts in the draft genome presents the research community with a new stream of data that will facilitate accurate determination of gene structures, genome annotation, and exploration of the transcriptome and mRNA metabolism in C. reinhardtii.

Extensive variation between inbred mouse strains due to endogenous L1 retrotransposition

Numerous inbred mouse strains comprise models for human diseases and diversity, but the molecular differences between them are mostly unknown. Several mammalian genomes have been assembled, providing a framework for identifying structural variations. To identify variants between inbred mouse strains at a single nucleotide resolution, we aligned 26 million individual sequence traces from four laboratory mouse strains to the C57BL/6J reference genome. We discovered and analyzed over 10,000 intermediate-length genomic variants (from 100 nucleotides to 10 kilobases), distinguishing these strains from the C57BL/6J reference. Approximately 85% of such variants are due to recent mobilization of endogenous retrotransposons, predominantly L1 elements, greatly exceeding that reported in humans. Many genes' structures and expression are altered directly by polymorphic L1 retrotransposons, including Drosha (also called Rnasen), Parp8, Scn1a, Arhgap15, and others, including novel genes. L1 polymorphisms are distributed nonrandomly across the genome, as they are excluded significantly from the X chromosome and from genes associated with the cell cycle, but are enriched in receptor genes. Thus, recent endogenous L1 retrotransposition has diversified genomic structures and transcripts extensively, distinguishing mouse lineages and driving a major portion of natural genetic variation.