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Viruses Feb 2021Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the... (Comparative Study)
Comparative Study
Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.
Topics: Genome, Viral; Genomic Library; Humans; Limit of Detection; Metagenomics; Nucleic Acid Amplification Techniques; Viruses
PubMed: 33562285
DOI: 10.3390/v13020253 -
Nucleic Acids Research Dec 2004Single-stranded DNA or RNA libraries used in SELEX experiments usually include primer-annealing sequences for PCR amplification. In genomic SELEX, these fixed sequences...
Single-stranded DNA or RNA libraries used in SELEX experiments usually include primer-annealing sequences for PCR amplification. In genomic SELEX, these fixed sequences may form base pairs with the central genomic fragments and interfere with the binding of target molecules to the genomic sequences. In this study, a method has been developed to circumvent these artificial effects. Primer-annealing sequences are removed from the genomic library before selection with the target protein and are then regenerated to allow amplification of the selected genomic fragments. A key step in the regeneration of primer-annealing sequences is to employ thermal cycles of hybridization-extension, using the sequences from unselected pools as templates. The genomic library was derived from the bacteriophage fd, and the gene 5 protein (g5p) from the phage was used as a target protein. After four rounds of primer-free genomic SELEX, most cloned sequences overlapped at a segment within gene 6 of the viral genome. This sequence segment was pyrimidine-rich and contained no stable secondary structures. Compared with a neighboring genomic fragment, a representative sequence from the family of selected sequences had about 23-fold higher g5p-binding affinity. Results from primer-free genomic SELEX were compared with the results from two other genomic SELEX protocols.
Topics: Base Sequence; Binding Sites; DNA Primers; DNA-Binding Proteins; Directed Molecular Evolution; Genome, Viral; Genomic Library; Genomics; Nucleic Acid Amplification Techniques; Viral Proteins
PubMed: 15601993
DOI: 10.1093/nar/gnh179 -
Eukaryotic Cell Apr 2003
Review
Topics: Animals; Chromosome Mapping; Evolution, Molecular; Genome; Genomic Instability; Genomic Library; Host-Parasite Interactions; Humans; Molecular Biology; Oomycetes; Sequence Homology, Nucleic Acid
PubMed: 12684368
DOI: 10.1128/EC.2.2.191-199.2003 -
Nature Communications Feb 2017Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including...
Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers. Next-generation sequencing approaches and other STR assays rely on a limited number of PCR amplicons, typically in the tens. Here, we demonstrate STR-Seq, a next-generation sequencing technology that analyses over 2,000 STRs in parallel, and provides the accurate genotyping of microsatellites. STR-Seq employs in vitro CRISPR-Cas9-targeted fragmentation to produce specific DNA molecules covering the complete microsatellite sequence. Amplification-free library preparation provides single molecule sequences without unique molecular barcodes. STR-selective primers enable massively parallel, targeted sequencing of large STR sets. Overall, STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared with other microsatellite analysis approaches. With these new features, STR-Seq can identify a 0.1% minor genome fraction in a DNA mixture composed of different, unrelated samples.
Topics: Alleles; CRISPR-Cas Systems; Genomic Library; Haplotypes; High-Throughput Nucleotide Sequencing; Human Genome Project; Humans; Microsatellite Repeats; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 28169275
DOI: 10.1038/ncomms14291 -
PeerJ 2022Genome skimming is a popular method in plant phylogenomics that do not include a biased enrichment step, relying on random shallow sequencing of total genomic DNA. From...
BACKGROUND
Genome skimming is a popular method in plant phylogenomics that do not include a biased enrichment step, relying on random shallow sequencing of total genomic DNA. From these data the plastome is usually readily assembled and constitutes the bulk of phylogenetic information generated in these studies. Despite a few attempts to use genome skims to recover low copy nuclear loci for direct phylogenetic use, such endeavor remains neglected. Causes might include the trade-off between libraries with few reads and species with large genomes (., missing data caused by low coverage), but also might relate to the lack of pipelines for data assembling.
METHODS
A pipeline and its companion R package designed to automate the recovery of low copy nuclear markers from genome skimming libraries are presented. Additionally, a series of analyses aiming to evaluate the impact of key assembling parameters, reference selection and missing data are presented.
RESULTS
A substantial amount of putative low copy nuclear loci was assembled and proved useful to base phylogenetic inference across the libraries tested (4 to 11 times more data than previously assembled plastomes from the same libraries).
DISCUSSION
Critical aspects of assembling low copy nuclear markers from genome skims include the minimum coverage and depth of a sequence to be used. More stringent values of these parameters reduces the amount of assembled data and increases the relative amount of missing data, which can compromise phylogenetic inference, in turn relaxing the same parameters might increase sequence error. These issues are discussed in the text, and parameter tuning through multiple comparisons tracking their effects on support and congruence is highly recommended when using this pipeline. The skimmingLoci pipeline (https://github.com/mreginato/skimmingLoci) might stimulate the use of genome skims to recover nuclear loci for direct phylogenetic use, increasing the power of genome skimming data to resolve phylogenetic relationships, while reducing the amount of sequenced DNA that is commonly wasted.
Topics: Phylogeny; Genome, Plant; Sequence Analysis, DNA; DNA; Genomic Library
PubMed: 36523475
DOI: 10.7717/peerj.14525 -
The Journal of Cell Biology Jul 2000
Review
Topics: Animals; Biological Evolution; Caenorhabditis elegans; Cell Adhesion; Drosophila melanogaster; Genome; Genomic Library; Humans
PubMed: 10908592
DOI: 10.1083/jcb.150.2.f89 -
Journal of Applied Microbiology Oct 2011Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability... (Review)
Review
Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function-based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic-based screens.
Topics: Bacteria; Biotechnology; Enzymes; Genomic Library; Industrial Microbiology; Metagenomics; Seawater; Surface-Active Agents
PubMed: 21777355
DOI: 10.1111/j.1365-2672.2011.05106.x -
The Journal of Cell Biology Jul 2000
Review
Topics: Adult; Animals; Drosophila melanogaster; GTP-Binding Proteins; Genome; Genomic Library; Humans; Receptors, Cell Surface
PubMed: 10908591
DOI: 10.1083/jcb.150.2.f83 -
BMC Genomics Mar 2022A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the...
BACKGROUND
A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries.
RESULTS
Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription.
CONCLUSIONS
Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.
Topics: Databases, Genetic; Genomic Library; High-Throughput Nucleotide Sequencing; Quality Control; Transcription, Genetic
PubMed: 35255806
DOI: 10.1186/s12864-022-08352-8 -
BMC Plant Biology Jul 2016Genomic libraries of introgression lines (ILs) consist of collections of homozygous lines with a single chromosomal introgression from a donor genotype in a common,...
BACKGROUND
Genomic libraries of introgression lines (ILs) consist of collections of homozygous lines with a single chromosomal introgression from a donor genotype in a common, usually elite, genetic background, representing the whole donor genome in the full collection. Currently, the only available melon IL collection was generated using Piel de sapo (var. inodorus) as the recurrent background. ILs are not available in genetic backgrounds representing other important market class cultivars, such as the cantalupensis. The recent availability of genomic tools in melon, such as SNP collections and genetic maps, facilitates the development of such mapping populations.
RESULTS
We have developed a new genomic library of introgression lines from the Japanese cv. Ginsen Makuwa (var. makuwa) into the French Charentais-type cv. Vedrantais (var. cantalupensis) genetic background. In order to speed up the breeding program, we applied medium-throughput SNP genotyping with Sequenom MassARRAY technology in early backcross generations and High Resolution Melting in the final steps. The phenotyping of the backcross generations and of the final set of 27 ILs (averaging 1.3 introgressions/plant and covering nearly 100 % of the donor genome), in three environments, allowed the detection of stable QTLs for flowering and fruit quality traits, including some that affect fruit size in chromosomes 6 and 11, others that change fruit shape in chromosomes 7 and 11, others that change flesh color in chromosomes 2, 8 and 9, and still others that increase sucrose content and delay climacteric behavior in chromosomes 5 and 10.
CONCLUSIONS
A new melon IL collection in the Charentais genetic background has been developed. Genomic regions that consistently affect flowering and fruit quality traits have been identified, which demonstrates the suitability of this collection for dissecting complex traits in melon. Additionally, pre-breeding lines with new, commercially interesting phenotypes have been observed, including delayed climacteric ripening associated to higher sucrose levels, which is of great interest for Charentais cultivar breeding.
Topics: Cucumis melo; Fruit; Genomic Library; Genotype; Polymorphism, Single Nucleotide; Quantitative Trait Loci
PubMed: 27390934
DOI: 10.1186/s12870-016-0842-0