scholarly journals Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Axel Poulet ◽  
Maud Privat ◽  
Flora Ponelle ◽  
Sandrine Viala ◽  
Stephanie Decousus ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Familial Risk ◽  
454 Sequencing ◽  
Diagnostic Procedures ◽  
Amplicon Sequencing ◽  
Sequencing Data ◽  
Dideoxy Sequencing ◽  
Ideal Situation

Screening forBRCAmutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of theBRCAgenes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

scholarly journals Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasemin Guenay-Greunke ◽  
David A. Bohan ◽  
Michael Traugott ◽  
Corinna Wallinger
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Sequencing Depth ◽  
Sequencing Error ◽  
Sequencing Data ◽  
Large Sample ◽  
Sequencing Errors ◽  
Plant Feeding

AbstractHigh-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

scholarly journals Equivolumetric Protocol Generates Library Sizes Proportional to Total Microbial Load in 16S Amplicon Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Giuliano Netto Flores Cruz ◽  
Ana Paula Christoff ◽  
Luiz Felipe Valter de Oliveira
Keyword(s):  
16S Rrna ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Predictive Performance ◽  
Amplicon Sequencing ◽  
Microbial Load ◽  
Cumulative Probability ◽  
Sequencing Data ◽  
Probability Models ◽  
Order Of Magnitude

High-throughput sequencing of 16S rRNA amplicon has been extensively employed to perform microbiome characterization worldwide. As a culture-independent methodology, it has allowed high-level profiling of sample bacterial composition directly from samples. However, most studies are limited to information regarding relative bacterial abundances (sample proportions), ignoring scenarios in which sample microbe biomass can vary widely. Here, we use an equivolumetric protocol for 16S rRNA amplicon library preparation capable of generating Illumina sequencing data responsive to input DNA, recovering proportionality between observed read counts and absolute bacterial abundances within each sample. Under specified conditions, we show that the estimation of colony-forming units (CFU), the most common unit of bacterial abundance in classical microbiology, is challenged mostly by resolution and taxon-to-taxon variation. We propose Bayesian cumulative probability models to address such issues. Our results indicate that predictive errors vary consistently below one order of magnitude for total microbial load and abundance of observed bacteria. We also demonstrate our approach has the potential to generalize to previously unseen bacteria, but predictive performance is hampered by specific taxa of uncommon profile. Finally, it remains clear that high-throughput sequencing data are not inherently restricted to sample proportions only, and such technologies bear the potential to meet the working scales of traditional microbiology.

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

2016 ◽  
Vol 82 (15) ◽  
pp. 4757-4766 ◽  
Author(s):  
Caterina R. Giner ◽  
Irene Forn ◽  
Sarah Romac ◽  
Ramiro Logares ◽  
Colomban de Vargas ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
Relative Abundance ◽  
Ribosomal Dna ◽  
18S Rdna ◽  
High Throughput Sequencing ◽  
454 Sequencing ◽  
Epifluorescence Microscopy ◽  
Environmental Sequencing

ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

Efficient High-throughput Techniques for the Analysis of Disease-Resistant Plant Varieties and Detection of Food Adulteration

2021 ◽  
Vol 23 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Rafiq Ahmad Bhat ◽  
Deyue Yu ◽  
Javaid Akhter Bhat
Keyword(s):  
Multiplex Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Crop Improvement ◽  
Small Sample ◽  
Food Crops ◽  
Genomic Resources ◽  
Breeding Programmes ◽  
Next Generation Sequencing Ngs

Abstract: Over the past two decades, the advances in the next generation sequencing (NGS) platforms have led to the identification of numerous genes/QTLs at high-resolution for their potential use in crop improvement. The genomic resources generated through these high-throughput sequencing techniques have been efficiently used in screening of particular gene of interest particularly for numerous types of plant stresses and quality traits. Subsequently, the identified-markers linked to a particular trait have been used in marker-assisted backcross breeding (MABB) activities. Besides, these markers are also being used to catalogue the food crops for detection of adulteration to improve the quality of food. With the advancement of technologies, the genomic resources are originating with new markers; however, to use these markers efficiently in crop breeding, high-throughput techniques (HTT) such as multiplex PCR and capillary electrophoresis (CE) can be exploited. Robustness, ease of operation, good reproducibility and low cost are the main advantages of multiplex PCR and CE. The CE is capable of separating and characterizing proteins with simplicity, speed and small sample requirements. Keeping in view the availability of vast data generated through NGS techniques and development of numerous markers, there is a need to use these resources efficiently in crop improvement programmes. In summary, this review describes the use of molecular markers in the screening of resistance genes in breeding programmes and detection of adulterations in food crops using high-throughput techniques.

scholarly journals Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding

MycoKeys ◽  
2018 ◽  
Vol 39 ◽  
pp. 29-40 ◽  
Author(s):  
Sten Anslan ◽  
R. Henrik Nilsson ◽  
Christian Wurzbacher ◽  
Petr Baldrian ◽  
Leho Tedersoo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Computation Time ◽  
Potential Effect ◽  
Data Sets ◽  
Sequencing Data ◽  
Operational Taxonomic Units ◽  
High Throughput Sequencing Data ◽  
Recent Developments

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Improvements and impacts of GRCh38 human reference on high throughput sequencing data analysis

Genomics ◽  
2017 ◽  
Vol 109 (2) ◽  
pp. 83-90 ◽  
Author(s):  
Yan Guo ◽  
Yulin Dai ◽  
Hui Yu ◽  
Shilin Zhao ◽  
David C. Samuels ◽  
...  
Keyword(s):  
Data Analysis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Data ◽  
High Throughput Sequencing Data ◽  
Sequencing Data Analysis
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