355 research outputs found
The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing.
No full textHigh-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. Despite producing gigabases of sequence per run, these technologies still present obstacles in resequencing and de novo assembly applications due to biased or insufficient target sequence coverage. We present here a simple sample preparation method termed the "long march" that increases both contig lengths and target sequence coverage using high-throughput short-read technologies. By incorporating a Type IIS restriction enzyme recognition motif into the sequencing primer adapter, successive rounds of restriction enzyme cleavage and adapter ligation produce a set of nested sub-libraries from the initial amplicon library. Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension. We demonstrate the utility of the long march in resequencing of the Plasmodium falciparum transcriptome, where the number of genomic bases covered was increased by 39%, as well as in metagenomic analysis of a serum sample from a patient with hepatitis B virus (HBV)-related acute liver failure, where the number of HBV bases covered was increased by 42%. We also offer a theoretical optimization of the long march for de novo sequence assembly
Framework of the Alu Subfamily Evolution in the Platyrrhine Three-Family Clade of Cebidae, Callithrichidae, and Aotidae
No full textThe history of Alu retroposons has been choreographed by the systematic accumulation of inherited diagnostic nucleotide substitutions to form discrete subfamilies, each having a distinct nucleotide consensus sequence. The oldest subfamily, AluJ, gave rise to AluS after the split between Strepsirrhini and what would become Catarrhini and Platyrrhini. The AluS lineage gave rise to AluY in catarrhines and to AluTa in platyrrhines. Platyrrhine Alu subfamilies Ta7, Ta10, and Ta15 were assigned names based on a standardized nomenclature. However, with the subsequent intensification of whole genome sequencing (WGS), large scale analyses to characterize Alu subfamilies using the program COSEG identified entire lineages of subfamilies simultaneously. The first platyrrhine genome with WGS, the common marmoset (Callithrix jacchus; [caljac3]), resulted in Alu subfamily names sf0 to sf94 in an arbitrary order. Although easily resolved by alignment of the consensus sequences, this naming convention can become increasingly confusing as more genomes are independently analyzed. In this study, we reported Alu subfamily characterization for the platyrrhine three-family clade of Cebidae, Callithrichidae, and Aotidae. We investigated one species/genome from each recognized family of Callithrichidae and Aotidae and of both subfamilies (Cebinae and Saimiriinae) of the family Cebidae. Furthermore, we constructed a comprehensive network of Alu subfamily evolution within the three-family clade of platyrrhines to provide a working framework for future research. Alu expansion in the three-family clade has been dominated by AluTa15 and its derivatives
Genetic composition of laboratory stocks of the self-fertilizing fish Kryptolebias marmoratus: a valuable resource for experimental research.
No full textThe hermaphroditic Mangrove Killifish, Kryptolebias marmoratus, is the world's only vertebrate that routinely self-fertilizes. As such, highly inbred and presumably isogenic "clonal" lineages of this androdioecious species have long been maintained in several laboratories and used in a wide variety of experiments that require genetically uniform vertebrate specimens. Here we conduct a genetic inventory of essentially all laboratory stocks of the Mangrove Killifish held worldwide. At 32 microsatellite loci, these stocks proved to show extensive interline differentiation as well as some intraline variation, much of which can be attributed to post-origin de novo mutations and/or to the segregation of polymorphisms from wild progenitors. Our genetic findings also document that many of the surveyed laboratory strains are not what they have been labeled, apparently due to the rather frequent mishandling or unintended mixing of various laboratory stocks over the years. Our genetic inventory should help to clarify much of this confusion about the clonal identities and genetic relationships of laboratory lines, and thereby help to rejuvenate interest in K. marmoratus as a reliable vertebrate model for experimental research that requires or can capitalize upon "clonal" replicate specimens
DNA samples of all species examined in this study.
No full texta<p>Coriell Institute for Medical Research, 403 Haddon Avenue, Camden, NJ 08103, USA.</p>b<p>Duke Lemur Center (DLC), Duke University, Durham, NC 27708, USA.</p>c<p>Integrated Primate Biomaterials and Information Resource (IPBIR), <a href="http://ccr.coriell.org/Sections/Collections/" target="_blank">http://ccr.coriell.org/Sections/Collections/</a>.</p>d<p>Frozen Zoo, San Diego Zoo (SDFZ), <a href="http://conservationandscience.org" target="_blank">http://conservationandscience.org</a>.</p>e<p>Gene Bank of Primates (GBP), German Primate Center, Göttingen, Germany.</p>f<p>Batzer: Adenovirus 12 SV40-transformed fibroblasts maintained in the lab of Dr. Mark Batzer.</p>g<p>From cell lines provided by American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA.</p><p>DNA samples of all species examined in this study.</p
Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.
No full textBackgroundHumans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).Methodology/principal findingsWe have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.ConclusionsWe report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described
Owl Monkey Alu Insertion Polymorphisms and Aotus Phylogenetics
No full textOwl monkeys (genus Aotus), or “night monkeys” are platyrrhine primates in the Aotidae family. Early taxonomy only recognized one species, Aotus trivirgatus, until 1983, when Hershkovitz proposed nine unique species designations, classified into red-necked and gray-necked species groups based predominately on pelage coloration. Recent studies questioned this conventional separation of the genus and proposed designations based on the geographical location of wild populations. Alu retrotransposons are a class of mobile element insertion (MEI) widely used to study primate phylogenetics. A scaffold-level genome assembly for one Aotus species, Aotus nancymaae [Anan_2.0], facilitated large-scale ascertainment of nearly 2000 young lineage-specific Alu insertions. This study provides candidate oligonucleotides for locus-specific PCR assays for over 1350 of these elements. For 314 Alu elements across four taxa with multiple specimens, PCR analyses identified 159 insertion polymorphisms, including 21 grouping A. nancymaae and Aotus azarae (red-necked species) as sister taxa, with Aotus vociferans and A. trivirgatus (gray-necked) being more basal. DNA sequencing identified five novel Alu elements from three different taxa. The Alu datasets reported in this study will assist in species identification and provide a valuable resource for Aotus phylogenetics, population genetics and conservation strategies when applied to wild populations
ACKNOWLEDGEMENTS
No full textI would like to thank my major professor, Dr. Mark A. Batzer for his remarkable mentorship and financial support to my studies and my dissertation committee member
Correction: An -Based Phylogeny of Lemurs (Infraorder: Lemuriformes).
No full textLEMURS (INFRAORDER: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55-60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of Eulemur and Varecia, with Varecia the sister lineage to the other three genera
Centromere remodeling in Hoolock leuconedys (Hylobatidae) by a new transposable element unique to the gibbons
No full textGibbons (Hylobatidae) shared a common ancestor with the other hominoids only 15-18 million years ago. Nevertheless, gibbons show very distinctive features that include heavily rearranged chromosomes. Previous observations indicate that this phenomenon may be linked to the attenuated epigenetic repression of transposable elements (TEs) in gibbon species.Here we describe the massive expansion of a repeat in almost all the centromeres of the Eastern hoolock gibbon (Hoolock leuconedys). We discovered that this repeat is a new composite TE originating from the combination of portions of three other elements (L1ME5, AluSz6, and SVA_A) and thus named it LAVA. We determined that this repeat is found in all the gibbons but does not occur in other hominoids. Detailed investigation of 46 different LAVA elements revealed that the majority of them have target site duplications (TSDs) and a poly-A tail, suggesting that they have been retrotransposing in the gibbon genome.Although we did not find a direct correlation between the emergence of LAVA elements and human-gibbon synteny breakpoints, this new composite transposable element is another mark of the great plasticity of the gibbon genome. Moreover, the centromeric expansion of LAVA insertions in the hoolock closely resembles the massive centromeric expansion of the KERV-1 retroelement reported for wallaby (marsupial) interspecific hybrids. The similarity between the two phenomena is consistent with the hypothesis that evolution of the gibbons was characterized by defects in epigenetic repression of TEs, perhaps triggered by interspecific hybridization
Sooty blotch and flyspeck on apple: expansion of the fungal complex, post-harvest removal and heterogeneity of apple canopy wetness and its impact on the outcome of a disease-warning system
No full textSooty blotch and flyspeck (SBFS) fungi blemish the cuticle of apples. Previous studies reported that the SBFS complex is comprised of four species. This is study surveyed the SBFS complex from nine orchards in four Midwestern states (USA). The LSU analyses of the rDNA inferred that 30 species were Dothideomycetes; one species was within the Pleosporales, 27 were within Dothideales, and two species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella. Post-harvest dips in commercial disinfestants were used to remove SBFS signs. Apples were dipped for 7 or 15 min in various concentrations of sodium hypochlorite, hydrogen peroxide and peroxyacetic acid mixtures, or soap, then brushed and rinsed on a grading line. A 7-min dip in 800 ppm chlorine resulted in an increase from 25% and 55% to 100% "Extra Fancy" grade for 'Jonathan' and 'Golden Delicious' apples, respectively, and increased market value by 31 and 14%, respectively. Blemishes were removed more effectively from 'Jonathan' and 'Macintosh' apples than from 'Golden Delicious'. SBFS fungi were removed differentially by the dip treatments. Leaf wetness duration (LWD) was measured within apple tree canopies in four Iowa orchards. Variability of LWD and the timing of dew onset and dry-off were characterized for twelve positions in the canopy of trees. The upper and eastern portion of the canopy was the first to form dew and the last to dry. The lower, western portion of the canopy usually averaged about 2 hours of LWD per day less than the top of the canopy, and was the last zone where dew formed and the first to dry off. When LWD were input to a warning system for the SBFS complex, timing of fungicide-spray thresholds varied by as much as 30 days among canopy positions.</p
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