scholarly journals Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Filipe Alves ◽  
Filipa M. S. Martins ◽  
Miguel Areias ◽  
Antonio Muñoz-Mérida
Keyword(s):  
High Throughput Sequencing ◽  
Population Studies ◽  
Population Level ◽  
Distribution Patterns ◽  
Pcr Primers ◽  
Population Diversity ◽  
Variable Regions ◽  
Polymorphic Loci ◽  
Population Extinction ◽  
Selection Of

AbstractAnalysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.

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.

scholarly journals Optimized genotyping method for identification of bacterial contaminants in pharmaceutical industry

2016 ◽  
Vol 66 (2) ◽  
pp. 289-295
Author(s):  
Borche Stamatoski ◽  
Miroslava Ilievska ◽  
Hristina Babunovska ◽  
Nikola Sekulovski ◽  
Sasho Panov
Keyword(s):  
Pharmaceutical Industry ◽  
Dna Sequencing ◽  
Bacterial Contamination ◽  
Raw Materials ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Bacterial Contaminants ◽  
Microbiological Control ◽  
Selection Of

AbstractMicrobiological control is of crucial importance in the pharmaceutical industry regarding the possible bacterial contamination of the environment, water, raw materials and finished products. Molecular identification of bacterial contaminants based on DNA sequencing of the hypervariable 16SrRNA gene has been introduced recently. The aim of this study is to investigate the suitability of gene sequencing using our selection of PCR primers and conditions for rapid and accurate bacterial identification in pharmaceutical industry quality control.DNA was extracted from overnight incubated colonies from 10 bacterial ATCC strains, which are common contaminants in the pharmaceutical industry. A region of bacterial 16SrRNA gene was analyzed by bidirectional DNA sequencing. Bacterial identification based on partial sequencing of the 16SrRNA gene is the appropriate method that could be used in the pharmaceutical industry after adequate validations. We have successfully identified all tested bacteria with more than 99 % similarity to the already published sequences.

Population-specific genome graphs improve high-throughput sequencing data analysis: A case study on the Pan-African genome

2021 ◽  
Author(s):  
H. Serhat Tetikol ◽  
Kubra Narci ◽  
Deniz Turgut ◽  
Gungor Budak ◽  
Ozem Kalay ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Information Overload ◽  
African Ancestry ◽  
Sample Selection ◽  
Variant Calling ◽  
Population Diversity ◽  
Human Populations ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Graph Augmentation

ABSTRACTGraph-based genome reference representations have seen significant development, motivated by the inadequacy of the current human genome reference for capturing the diverse genetic information from different human populations and its inability to maintain the same level of accuracy for non-European ancestries. While there have been many efforts to develop computationally efficient graph-based bioinformatics toolkits, how to curate genomic variants and subsequently construct genome graphs remains an understudied problem that inevitably determines the effectiveness of the end-to-end bioinformatics pipeline. In this study, we discuss major obstacles encountered during graph construction and propose methods for sample selection based on population diversity, graph augmentation with structural variants and resolution of graph reference ambiguity caused by information overload. Moreover, we present the case for iteratively augmenting tailored genome graphs for targeted populations and test the proposed approach on the whole-genome samples of African ancestry. Our results show that, as more representative alternatives to linear or generic graph references, population-specific graphs can achieve significantly lower read mapping errors, increased variant calling sensitivity and provide the improvements of joint variant calling without the need of computationally intensive post-processing steps.

scholarly journals Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood

2014 ◽  
Author(s):  
Fan Gao ◽  
Kai Wang
Keyword(s):  
High Throughput Sequencing ◽  
Read Length ◽  
Integrative Approach ◽  
Sequencing Data ◽  
Immune Repertoire ◽  
Universal Primer ◽  
Illumina Hiseq ◽  
Variable Regions ◽  
Anchored Pcr ◽  
Pcr Method

Background As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR method has been previously developed to assay immune repertoire, however the usage of primer pools has inherent bias in target amplification. In our study, we developed a ligation-anchored PCR method to unbiasedly amplify the repertoire. Results By utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which provided 151 bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from patients with malignant and benign meningiomas. Mapping of sequencing data showed 64% to 91% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of TRBV29-1 was observed in malignant meningiomas. Also distinct signatures were identified from CDR3 sequence logos, with predominant subset as 42 nt for benign and 45 nt for malignant samples, respectively. Conclusions In summary, we report an integrative approach to monitor immune repertoire in a systematic manner.

scholarly journals KERAGAMAN GENETIK MUTAN M-2 CABAI MERAH KERITING (Capsicum annuum L.) BERDASARKAN PENANDA RAPD

2020 ◽  
Vol 10 (2) ◽  
pp. 92
Author(s):  
Rosmaina Rosmaina ◽  
Dedi Mulyadi ◽  
Rita Elfianis ◽  
Zulfahmi Zulfahmi
Keyword(s):  
Capsicum Annuum ◽  
Gamma Ray ◽  
Rapd Analysis ◽  
Genetic Material ◽  
Polymorphic Loci ◽  
Capsicum Annuum L ◽  
Horticultural Plant ◽  
Dna Fragment ◽  
And Control ◽  
Selection Of

Chili is an important horticultural plant in Indonesia. This research aims to carry out RAPD analysis on Mutant M2 of chili pepper (Capsicum annuum L.). Six M2 genotypes of chili irradiated by gamma ray and control plants were amplified by 16 random primers. The amplification results of M2 chili with 16 primers produced 118 loci, with fragment sizes ranging from 150-2000 bp. The number of polymorphic loci was 96 loci and the percentage of polymorphic loci was 83.23%. The DNA fragment polymorphism produced in this research was relatively high and it showed that the gamma ray mutagen applied produced high chili genetic diversity. The value of genetic similarity between control plants and mutant plants ranged from 0.7474 to 0.4874. UPGMA dendogram classified seven genotypes tested into three groups, the first group consisted of mutants R2U6 and R2U17, the second group was mutants R1U14 and R1U17, and the third group was mutants R2U8, mutants R2U2 and control plants. The finding of this research can be used as a basic selection of genetic material for chili’s breeding in the future.

scholarly journals Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome

2015 ◽  
Vol 212 (10) ◽  
pp. 1663-1677 ◽  
Author(s):  
Nikita S. Kolhatkar ◽  
Archana Brahmandam ◽  
Christopher D. Thouvenel ◽  
Shirly Becker-Herman ◽  
Holly M. Jacobs ◽  
...  
Keyword(s):  
B Cells ◽  
Positive Selection ◽  
B Cell ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Cell Stage ◽  
Wiskott Aldrich Syndrome ◽  
Transitional B Cells ◽  
Selection Of

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias. WAS protein (WASp)–deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire. To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12). High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells. Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals. Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects. Our combined data support a model wherein modest alterations in B cell–intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells.

Molecular characterization and structure of intestinal micro flora for postmortem interval estimation in SD rats

2019 ◽  
Author(s):  
Huan Li ◽  
Lu Yuan ◽  
Ruina Liu ◽  
Siruo Zhang ◽  
E Yang ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput Sequencing ◽  
Interval Estimation ◽  
Pcr Amplification ◽  
Postmortem Interval ◽  
The Body ◽  
Rrna Gene ◽  
Variable Regions ◽  
Time Points ◽  
Almost All

Abstract Background The human rectum flora consists of a huge variety of bacteria and the association between individuals and their rectum bacterial community begins presently after birth and continues the whole lifetime. Once the body dies, the inherent microbes begin to break down from the inside and play a key role thereafter. Results The aim of this study was to investigate the probable shift of the rectum flora at different time intervals up to 15 days after death and to characterize the contribution for of this shift to estimate the time of death. The rectum of rats was wiped with a sterile cotton swab and the samples were proceeded for DNA extraction, PCR amplification of the 16S rRNA gene with the V3+V4 variable regions, and high throughput sequencing carried out on IonS5TMXL platform. The results were analyzed for intra-group and inter-group diversity, similarity and difference at different time points. At phylum level, Proteobacteria and Firmicutes showed major shifts, checked at 11 different intervals and emerged in the most of postmortem intervals. At the genus level, Enterococcus appeared in all groups except alive samples, Lactobacillus and Proteus appeared in most time points, and the latter showed an increasing trend after 3 days postmortem samples. At the species level, Enterococcus_faecalis and Proteus_mirabilis existed in most postmortem intervals, and the former had a downward trend after day 5 postmortem, while the latter had an upward trend. Corynebacterium_amycolatum , Entero_isolate_group_2 , Bacteroides_uniformis , Enterococcus_faecalis , Streptococcus_gallolyticus_subsp_macedonics , Clostridium_sporogenes were more abundant in 0-hour, day 1, 3, 5, 7, 13 postmortem intervals, respectively, while Proteus_mirabilis and Vagococcus_lutrae were abundant in day 15 postmortem. In addition, functional capacity analysis of Membrane_Transport, Amino_Acid_Metabolism, Nucleotide_Metabolism and Energy_Metabolism showed significant differences between alive and almost all other time points after death ( P <0.05). Conclusions All in all, bacteria at different levels (phylum, genera, species) showed different characteristic during the process of decomposition and possessed entirely different relative abundance and the structure of bacterial community in each time point shifted obviously, which suggested that the specific bacteria might imply the specific postmortem interval during decomposition.

scholarly journals Chinese Fir Breeding in the High-Throughput Sequencing Era: Insights from SNPs

Forests ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 681 ◽  
Author(s):  
Huiquan Zheng ◽  
Dehuo Hu ◽  
Ruping Wei ◽  
Shu Yan ◽  
Runhui Wang
Keyword(s):  
Population Structure ◽  
Genetic Structure ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Breeding Population ◽  
Population Diversity ◽  
Chinese Fir ◽  
High Density ◽  
Population Structure Analysis ◽  
Snp Panel

Knowledge on population diversity and structure is of fundamental importance for conifer breeding programs. In this study, we concentrated on the development and application of high-density single nucleotide polymorphism (SNP) markers through a high-throughput sequencing technique termed as specific-locus amplified fragment sequencing (SLAF-seq) for the economically important conifer tree species, Chinese fir (Cunninghamia lanceolata). Based on the SLAF-seq, we successfully established a high-density SNP panel consisting of 108,753 genomic SNPs from Chinese fir. This SNP panel facilitated us in gaining insight into the genetic base of the Chinese fir advance breeding population with 221 genotypes for its genetic variation, relationship and diversity, and population structure status. Overall, the present population appears to have considerable genetic variability. Most (94.15%) of the variability was attributed to the genetic differentiation of genotypes, very limited (5.85%) variation occurred on the population (sub-origin set) level. Correspondingly, low FST (0.0285–0.0990) values were seen for the sub-origin sets. When viewing the genetic structure of the population regardless of its sub-origin set feature, the present SNP data opened a new population picture where the advanced Chinese fir breeding population could be divided into four genetic sets, as evidenced by phylogenetic tree and population structure analysis results, albeit some difference in membership of the corresponding set (cluster vs. group). It also suggested that all the genetic sets were admixed clades revealing a complex relationship of the genotypes of this population. With a step wise pruning procedure, we captured a core collection (core 0.650) harboring 143 genotypes that maintains all the allele, diversity, and specific genetic structure of the whole population. This generalist core is valuable for the Chinese fir advanced breeding program and further genetic/genomic studies.

The sponge genetree server—providing a phylogenetic backbone for poriferan evolutionary studies

Zootaxa ◽  
2008 ◽  
Vol 1939 (1) ◽  
pp. 58-60 ◽  
Author(s):  
DIRK ERPENBECK ◽  
OLIVER VOIGT ◽  
MEHMET GÜLTAS ◽  
GERT WÖRHEIDE
Keyword(s):  
Phylogenetic Relationships ◽  
Population Studies ◽  
Phylogenetic Signal ◽  
Careful Evaluation ◽  
Evolutionary Studies ◽  
Individual Project ◽  
Dna Sequence Markers ◽  
Species Assignment ◽  
Definition Of ◽  
Selection Of

Unravelling the phylogenetic relationships of sponges (Phylum Porifera) is an important as well as challenging task. It helps the understanding of character evolution among early branching metazoans but also aids in bioprospecting for valuable bioactive sponge compounds. However, the phylogenetic relationships among Porifera are largely unsolved, because the simple poriferan bauplan frequently prevents unambiguous taxonomic species assignment and a clear definition of morphological synapomorphies is difficult (see e.g. Boury-Esnault 2006). DNA sequence markers are frequently employed to overcome morphological shortcomings in phylogeny (e.g. Kelly Borges et al. 1991) and taxonomy (e.g. DNA barcoding, see Wörheide & Erpenbeck 2007). However, some DNA markers suffer from insufficient phylogenetic signal (see e.g. Duran et al. 2004 and Wörheide 2006 on CO1 in population studies) and unequal evolutionary rates among taxa (see e.g. Erpenbeck et al. 2004 on 28S in Haplosclerida). Therefore, a careful evaluation and selection of molecular markers for each individual project is required.

scholarly journals Dataset of Targeted Metabolite Analysis for Five Taxanes of Hellenic Taxus baccata L. Populations

Data ◽  
2020 ◽  
Vol 5 (1) ◽  
pp. 22
Author(s):  
Eleftheria Dalmaris ◽  
Evangelia V. Avramidou ◽  
Aliki Xanthopoulou ◽  
Filippos A. Aravanopoulos
Keyword(s):  
Natural Populations ◽  
Population Level ◽  
Primary Sources ◽  
Taxus Baccata ◽  
Metabolite Analysis ◽  
Data Set ◽  
Baccatin Iii ◽  
Significant Seasonal Variation ◽  
Increasing Demand ◽  
Selection Of

Novel primary sources of one of the world’s leading anticancer agent, paclitaxel, as well as of other antineoplastic taxanes such as 10-deacetylbaccatin-III, are needed to meet an increasing demand. Among the Taxus species the promise of Taxus baccata L. (European or English yew) has been documented. In this study, the metabolite analysis of two marginal T. baccata populations in Greece (Mt. Cholomon and Mt. Olympus), located at the southeastern edge of the species natural distribution, are being explored. A targeted liquid chromatography – mass spectrometry (LC-MS/MS) analysis was used to determine the content of 10-deacetylbaccatin III, baccatin III, 10-deacetyltaxol, paclitaxel and cephalomannine in the needles of each of the populations from three sampling periods (spring, summer and winter). This is the first survey to generate a taxane targeted metabolite data set, since it derives from Hellenic natural populations that have not been explored before. Furthermore, it has used an extensive sample design in order to evaluate chemodiversity at the population level. The analysis revealed significant levels of chemodiversity within and among the investigated populations and significant seasonal variation that could be exploited for the selection of superior germplasm native to Greece, for yew plantations and further exploitation which is necessary for the production of important taxanes.

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