The uniqueome: a mappability resource for short-tag sequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Koehler R
PROVIDER: S-EPMC3018812 | biostudies-other | 2011 Jan
REPOSITORIES: biostudies-other
Publications
The uniqueome: a mappability resource for short-tag sequencing.
Koehler Ryan R Issac Hadar H Cloonan Nicole N Grimmond Sean M SM
Bioinformatics (Oxford, England) 20101112 2
<h4>Summary</h4>Quantification applications of short-tag sequencing data (such as CNVseq and RNAseq) depend on knowing the uniqueness of specific genomic regions at a given threshold of error. Here, we present the 'uniqueome', a genomic resource for understanding the uniquely mappable proportion of genomic sequences. Pre-computed data are available for human, mouse, fly and worm genomes in both color-space and nucletotide-space, and we demonstrate the utility of this resource as applied to the q ...[more]
Similar Datasets
Project description:RNA sequencing has become widely used in gene expression profiling experiments. Prior to any RNA sequencing experiment the quality of the RNA must be measured to assess whether or not it can be used for further downstream analysis. The RNA integrity number (RIN) is a scale used to measure the quality of RNA that runs from 1 (completely degraded) to 10 (intact). Ideally, samples with high RIN (> 8) are used in RNA sequencing experiments. RNA, however, is a fragile molecule which is susceptible to degradation and obtaining high quality RNA is often hard, or even impossible when extracting RNA from certain clinical tissues. Thus, occasionally, working with low quality RNA is the only option the researcher has. Here we investigate the effects of RIN on RNA sequencing and suggest a computational method to handle data from samples with low quality RNA which also enables reanalysis of published datasets. Using RNA from a human cell line we generated and sequenced samples with varying RINs and illustrate what effect the RIN has on the basic procedure of RNA sequencing; both quality aspects and differential expression. We show that the RIN has systematic effects on gene coverage, false positives in differential expression and the quantification of duplicate reads. We introduce 3' tag counting (3TC) as a computational approach to reliably estimate differential expression for samples with low RIN. We show that using the 3TC method in differential expression analysis significantly reduces false positives when comparing samples with different RIN, while retaining reasonable sensitivity.
| S-EPMC3954844 | biostudies-literature
Project description:We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.
| S-EPMC2848820 | biostudies-literature
Project description:Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ? 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ? 0.005% to ? 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.
| S-EPMC4026136 | biostudies-literature
Project description:Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6-V8, and V7-V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.
| S-EPMC4523815 | biostudies-literature
Project description:Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.
| S-EPMC3979301 | biostudies-other
Project description:BackgroundButterfly wing color patterns are a key model for integrating evolutionary developmental biology and the study of adaptive morphological evolution. Yet, despite the biological, economical and educational value of butterflies they are still relatively under-represented in terms of available genomic resources. Here, we describe an Expression Sequence Tag (EST) project for Bicyclus anynana that has identified the largest available collection to date of expressed genes for any butterfly.ResultsBy targeting cDNAs from developing wings at the stages when pattern is specified, we biased gene discovery towards genes potentially involved in pattern formation. Assembly of 9,903 ESTs from a subtracted library allowed us to identify 4,251 genes of which 2,461 were annotated based on BLAST analyses against relevant gene collections. Gene prediction software identified 2,202 peptides, of which 215 longer than 100 amino acids had no homology to any known proteins and, thus, potentially represent novel or highly diverged butterfly genes. We combined gene and Single Nucleotide Polymorphism (SNP) identification by constructing cDNA libraries from pools of outbred individuals, and by sequencing clones from the 3' end to maximize alignment depth. Alignments of multi-member contigs allowed us to identify over 14,000 putative SNPs, with 316 genes having at least one high confidence double-hit SNP. We furthermore identified 320 microsatellites in transcribed genes that can potentially be used as genetic markers.ConclusionOur project was designed to combine gene and sequence polymorphism discovery and has generated the largest gene collection available for any butterfly and many potential markers in expressed genes. These resources will be invaluable for exploring the potential of B. anynana in particular, and butterflies in general, as models in ecological, evolutionary, and developmental genetics.
| S-EPMC1534037 | biostudies-literature
Project description:Chromatin Interaction Analysis Using Paired-End Tag Sequencing (ChIA-PET) is an established method to map protein-mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is impractical to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA-PET protocol, which requires as few as 1 million cells. Here, we describe detailed step-by-step procedures for performing in situ ChIA-PET from cultured cells, including both an experimental protocol for sample preparation and data generation and a computational protocol for data processing and visualization using the ChIA-PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces ligation noise, and decreases the required input of cells compared to previous versions of ChIA-PET protocols, it can be applied to generate high-resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and data generation Support Protocol: Bridge linker preparation Basic Protocol 2: Data processing and visualization.
| S-EPMC8351913 | biostudies-literature
Project description:Analysis of expressed sequence tags (ESTs) constitutes a useful approach for gene identification that, in the case of human pathogens, might result in the identification of new targets for chemotherapy and vaccine development. As part of the Trypanosoma cruzi genome project, we have partially sequenced the 5' ends of 1, 949 clones to generate ESTs. The clones were randomly selected from a normalized CL Brener epimastigote cDNA library. A total of 14.6% of the clones were homologous to previously identified T. cruzi genes, while 18.4% had significant matches to genes from other organisms in the database. A total of 67% of the ESTs had no matches in the database, and thus, some of them might be T. cruzi-specific genes. Functional groups of those sequences with matches in the database were constructed according to their putative biological functions. The two largest categories were protein synthesis (23.3%) and cell surface molecules (10.8%). The information reported in this paper should be useful for researchers in the field to analyze genes and proteins of their own interest.
| S-EPMC108675 | biostudies-literature
Project description:Nitrogen can greatly influence the structure and productivity of microbial communities through its relative availability and form. However, the roles of specific organisms in the uptake of different nitrogen species remain poorly characterized. Most studies seeking to identify agents of assimilation have been correlative, indirectly linking activity measurements (e.g., nitrate uptake) with the presence or absence of biological markers, particularly functional genes and their transcripts. Evidence is accumulating of previously underappreciated functional diversity in major microbial subpopulations, which may confer physiological advantages under certain environmental conditions leading to ecotype divergence. This microdiversity further complicates our view of genetic variation in environmental samples requiring the development of more targeted approaches. Here, next-generation tag sequencing was successfully coupled with stable isotope probing (Tag-SIP) to assess the ability of individual phylotypes to assimilate a specific N source. Our results provide the first direct evidence of nitrate utilization by organisms thought to lack the genes required for this process including the heterotrophic clades SAR11 and the Archaeal Marine Group II. Alternatively, this may suggest the existence of tightly coupled metabolisms with primary assimilators, e.g., symbiosis, or the rapid and efficient scavenging of recently released products by highly active individuals. These results may be connected with global dominance often seen with these clades, likely conferring an advantage over other clades unable to access these resources. We also provide new direct evidence of in situ nitrate utilization by the cyanobacterium Prochlorococcus in support of recent findings. Furthermore, these results revealed widespread functional heterogeneity, i.e., different levels of nitrogen assimilation within clades, likely reflecting niche partitioning by ecotypes.
| S-EPMC5133248 | biostudies-literature
Project description:The present study was designed to explore the possibility of improving lipid production in oleaginous filamentous fungus Mortierella alpina based on an autophagy regulation strategy. According to multiomics information, vacuolate-centered macroautophagy was identified as the main type of autophagy in M. alpina under nitrogen-limited conditions. Mutation of autophagy-related gene MAatg8 led to impaired fatty acid synthesis, while overexpression of both MAatg8 and phosphatidylserine decarboxylases (MApsd2) showed promoting effects on fatty acid synthesis. MAatg8 overexpression strain with external supply of ethanolamine significantly increased arachidonic acid (ARA)-rich triacylglycerol (TAG) and biomass synthesis in M. alpina, and the final fatty acid content increased by approximately 110% compared with that in the wild-type strain. Metabolomics and lipidomics analyses revealed that cell autophagy enhanced the recycling of preformed carbon, nitrogen, and lipid in mycelium, and the released carbon skeleton and energy were contributed to the accumulation of TAG in M. alpina. This study suggests that regulation of autophagy-related MAatg8-phosphatidylethanolamine (MAatg8-PE) conjugation system could be a promising strategy for attaining higher lipid production and biomass growth. The mechanism of autophagy in regulating nitrogen limitation-induced lipid accumulation elucidated in this study provides a reference for development of autophagy-based strategies for improving nutrient use efficiency and high value-added lipid production by oleaginous microorganism. IMPORTANCE Studies have indicated that functional oil accumulation occurs in oleaginous microorganisms under nitrogen limitation. However, until now, large-scale application of nitrogen-deficiency strategies was limited by low biomass. Therefore, the identification of the critical nodes of nitrogen deficiency-induced lipid accumulation is urgently needed to further guide functional oil production. The significance of our research is in uncovering the function of cell autophagy in the ARA-rich TAG accumulation of oleaginous fungus M. alpina and demonstrating the feasibility of improving lipid production based on an autophagy regulation strategy at the molecular and omics levels. Our study proves that regulation of cell autophagy through the MAatg8-PE conjugation system-related gene overexpression or exogenous supply of ethanolamine would be an efficient strategy to increase and maintain biomass productivity when high TAG content is obtained under nitrogen deficiency, which could be useful for the development of new strategies that will achieve more biomass and maximal lipid productivity.
| S-EPMC8881083 | biostudies-literature