scholarly journals FecalSeq: methylation-based enrichment for noninvasive population genomics from feces

2015 ◽  
Author(s):  
Kenneth L. Chiou ◽  
Christina M. Bergey
Keyword(s):  
High Throughput Sequencing ◽  
Population Genomics ◽  
Population Level ◽  
Cost Effective ◽  
Fecal Dna ◽  
Host Animal ◽  
Preparation Methods ◽  
Exogenous Dna ◽  
Next Generation Sequencing Technology ◽  
Genomic Scale

AbstractObtaining high-quality samples from wild animals is a major obstacle for genomic studies of many taxa, particular at the population level, as collection methods for such samples are typically invasive. DNA from feces is easy to obtain noninvasively, but is dominated by a preponderance of bacterial and other non-host DNA. Because next-generation sequencing technology sequences DNA largely indiscriminately, the high proportion of exogenous DNA drastically reduces the efficiency of high-throughput sequencing for host animal genomics. In order to address this issue, we developed an inexpensive methylation-based capture method for enriching host DNA from noninvasively obtained fecal DNA samples. Our method exploits natural differences in CpG-methylation density between vertebrate and bacterial genomes to preferentially bind and isolate host DNA from majority-bacterial fecal DNA samples. We demonstrate that the enrichment is robust, efficient, and compatible with downstream library preparation methods useful for population studies (e.g., RADseq). Compared to other enrichment strategies, our method is quick and inexpensive, adding only a negligible cost to sample preparation for research that is often severely constrained by budgetary limitations. In combination with downstream methods such as RADseq, our approach allows for cost-effective and customizable genomic-scale genotyping that was previously feasible in practice only with invasive samples. Because feces are widely available and convenient to collect, our method empowers researchers to explore genomic-scale population-level questions in organisms for which invasive sampling is challenging or undesirable.

scholarly journals An evaluation of pool-sequencing transcriptome-based exon capture for population genomics in non-model species

2019 ◽  
Author(s):  
Emeline Deleury ◽  
Thomas Guillemaud ◽  
Aurélie Blin ◽  
Eric Lombaert
Keyword(s):  
Evolutionary Biology ◽  
High Throughput Sequencing ◽  
Harmonia Axyridis ◽  
Population Genomics ◽  
De Novo ◽  
Cost Effective ◽  
Allele Frequencies ◽  
Technical Solution ◽  
Model Species ◽  
Exon Capture

AbstractExon capture coupled to high-throughput sequencing constitutes a cost-effective technical solution for addressing specific questions in evolutionary biology by focusing on expressed regions of the genome preferentially targeted by selection. Transcriptome-based capture, a process that can be used to capture the exons of non-model species, is use in phylogenomics. However, its use in population genomics remains rare due to the high costs of sequencing large numbers of indexed individuals across multiple populations. We evaluated the feasibility of combining transcriptome-based capture and the pooling of tissues from numerous individuals for DNA extraction as a cost-effective, generic and robust approach to estimating the variant allele frequencies of any species at the population level. We designed capture probes for ∼5 Mb of chosen de novo transcripts from the Asian ladybird Harmonia axyridis (5,717 transcripts). We called ∼300,000 bi-allelic SNPs for a pool of 36 non-indexed individuals. Capture efficiency was high, and pool-seq was as effective and accurate as individual-seq for detecting variants and estimating allele frequencies. Finally, we also evaluated an approach for simplifying bioinformatic analyses by mapping genomic reads directly to targeted transcript sequences to obtain coding variants. This approach is effective and does not affect the estimation of SNP allele frequencies, except for a small bias close to some exon ends. We demonstrate that this approach can also be used to predict the intron-exon boundaries of targeted de novo transcripts, making it possible to abolish genotyping biases near exon ends.

Review on Methodologies Used in the Synthesis of Metal Nanoparticles: Significance of Phytosynthesis Using Plant Extract as an Emerging Tool

2020 ◽  
Vol 26 (40) ◽  
pp. 5188-5204
Author(s):  
Uzair Nagra ◽  
Maryam Shabbir ◽  
Muhammad Zaman ◽  
Asif Mahmood ◽  
Kashif Barkat
Keyword(s):  
Green Synthesis ◽  
Metal Nanoparticles ◽  
Plant Extracts ◽  
Biomedical Applications ◽  
Noble Metals ◽  
Cost Effective ◽  
Green Technology ◽  
Critical Parameters ◽  
Nanosized Particles ◽  
Preparation Methods

Nanosized particles, with a size of less than 100 nm, have a wide variety of applications in various fields of nanotechnology and biotechnology, especially in the pharmaceutical industry. Metal nanoparticles [MNPs] have been synthesized by different chemical and physical procedures. Still, the biological approach or green synthesis [phytosynthesis] is considered as a preferred method due to eco-friendliness, nontoxicity, and cost-effective production. Various plants and plant extracts have been used for the green synthesis of MNPs, including biofabrication of noble metals, metal oxides, and bimetallic combinations. Biomolecules and metabolites present in plant extracts cause the reduction of metal ions into nanosized particles by one-step preparation methods. MNPs have remarkable attractiveness in biomedical applications for their use as potential antioxidant, anticancer and antibacterial agents. The present review offers a comprehensive aspect of MNPs production via top-to-bottom and bottom-to-top approach with considerable emphasis on green technology and their possible biomedical applications. The critical parameters governing the MNPs formation by plant-based synthesis are also highlighted in this review.

scholarly journals INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5054
Author(s):  
Qianxin Wu ◽  
Chenqu Suo ◽  
Tom Brown ◽  
Tengyao Wang ◽  
Sarah A. Teichmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Population Level ◽  
Reaction Products ◽  
Testing Strategy ◽  
Asymptomatic Patients ◽  
Testing Stage ◽  
Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

Evaluation of the effectiveness of skin preparation methods for the reduction of Cutibacterium acnes (formerly Propionibacterium acnes) in shoulder surgery: a systematic review

2021 ◽  
pp. 175857322110325
Author(s):  
Maria Sagkrioti ◽  
Stephen Glass ◽  
Georgios Arealis
Keyword(s):  
Systematic Review ◽  
Hydrogen Peroxide ◽  
Side Effects ◽  
Cost Effective ◽  
Shoulder Surgery ◽  
Skin Preparation ◽  
Preparation Methods ◽  
Cutibacterium Acnes ◽  
Surgery Outcomes ◽  
Positive Effect

Background Cutibacterium acnes ( C. acnes) is the most common pathogen responsible for post-operative shoulder infections. The purpose of this study was to evaluate the effectiveness of skin preparation methods against C. acnes in shoulder surgery. Methods A systematic review was conducted evaluating the effectiveness of skin preparation methods in the reduction of C. acnes in patients undergoing shoulder surgery. Outcomes were assessed based on the effectiveness of the method used; side effects and cost were also analysed. Results Of the 19 included studies, 9 evaluated pre-surgical home treatments: 8 assessed benzoyl peroxide (BPO) and 6 concluded it is effective in reducing C. acnes. Nine studies assessed surgical skin preparation and concluded that Chlorhexidine gluconate (CHG) was not effective; in contrast hydrogen peroxide reduced C. acnes. Finally, one study evaluated an aseptic protocol using CHG and concluded that it was not effective. Conclusions It was demonstrated that BPO as home treatment is effective in reducing C. acnes load on skin ; it rarely causes side effects and is also cost-effective. This study highlights non-effectiveness of CHG. There was some evidence that the addition of hydrogen peroxide could have a positive effect in the reduction of C. acnes skin load; however, more studies are required.

scholarly journals TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1223
Author(s):  
Andres S. Espindola ◽  
Daniela Sempertegui-Bayas ◽  
Danny F. Bravo-Padilla ◽  
Viviana Freire-Zapata ◽  
Francisco Ochoa-Corona ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Rna Viruses ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Bioinformatic Analysis ◽  
Metagenomic Sample ◽  
Rrna Depletion ◽  
Virus Diagnostics ◽  
Management Preparation ◽  
Reverse Transcript

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

scholarly journals Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

2021 ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0247541
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms

2014 ◽  
Author(s):  
Jonathan Puritz ◽  
Christopher M. Hollenbeck ◽  
John R. Gold
Keyword(s):  
Population Genomics ◽  
De Novo ◽  
Variant Calling ◽  
Population Level ◽  
Model Organisms ◽  
Effective Population ◽  
Reduction Techniques ◽  
Indel Polymorphisms ◽  
Indel Calling ◽  
Population Sizes

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for organisms with large effective population sizes and high levels of genetic polymorphism but for which no genomic resources exist. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is most likely due to the fact that dDocent quality trims instead of filtering and incorporates both forward and reverse reads in assembly, mapping, and SNP calling, thus enabling use of reads with Indel polymorphisms. The pipeline and a comprehensive user guide can be found at (http://dDocent.wordpress.com).

scholarly journals Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology

2021 ◽  
Vol 111 (1) ◽  
pp. 8-11
Author(s):  
Remco Stam ◽  
Pierre Gladieux ◽  
Boris A. Vinatzer ◽  
Erica M. Goss ◽  
Neha Potnis ◽  
...  
Keyword(s):  
Population Genetics ◽  
Population Genomics ◽  
Cost Effective ◽  
Whole Genome ◽  
Genome Data ◽  
Sequencing Technologies ◽  
Population Genomic ◽  
High Throughput Dna Sequencing ◽  
Opening Up ◽  
Insight Into

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue . Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

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