A comparison of benchtop high-throughput sequencing platforms in the diagnostic laboratory setting

Pathology ◽  
2016 ◽  
Vol 48 ◽  
pp. S96
Author(s):  
Sarah L. Nickerson ◽  
Debra O. Prosser ◽  
Stella W.S. Lai ◽  
Donald R. Love
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Laboratory Setting ◽  
Diagnostic Laboratory ◽  
Sequencing Platforms

scholarly journals Technological advancements and their importance for nematode identification

SOIL ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 257-270 ◽  
Author(s):  
Mohammed Ahmed ◽  
Melanie Sapp ◽  
Thomas Prior ◽  
Gerrit Karssen ◽  
Matthew Alan Back
Keyword(s):  
High Throughput ◽  
Crop Production ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Biological Indicators ◽  
Rapid Identification ◽  
Community Studies ◽  
Terrestrial Environments ◽  
Traditional Taxonomy ◽  
Sequencing Platforms

Abstract. Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

Performance comparison of benchtop high-throughput sequencing platforms

2012 ◽  
Vol 30 (5) ◽  
pp. 434-439 ◽  
Author(s):  
Nicholas J Loman ◽  
Raju V Misra ◽  
Timothy J Dallman ◽  
Chrystala Constantinidou ◽  
Saheer E Gharbia ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Performance Comparison ◽  
Sequencing Platforms

scholarly journals Identification of Novel Viruses in Amblyomma americanum , Dermacentor variabilis , and Ixodes scapularis Ticks

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Rafal Tokarz ◽  
Stephen Sameroff ◽  
Teresa Tagliafierro ◽  
Komal Jain ◽  
Simon H. Williams ◽  
...  
Keyword(s):  
New York ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Ixodes Scapularis ◽  
The United States ◽  
Dermacentor Variabilis ◽  
Amblyomma Americanum ◽  
Animal Pathogens ◽  
Wide Range ◽  
Sequencing Platforms

ABSTRACT Ticks carry a wide range of known human and animal pathogens and are postulated to carry others with the potential to cause disease. Here we report a discovery effort wherein unbiased high-throughput sequencing was used to characterize the virome of 2,021 ticks, including Ixodes scapularis ( n = 1,138), Amblyomma americanum ( n = 720), and Dermacentor variabilis ( n = 163), collected in New York, Connecticut, and Virginia in 2015 and 2016. We identified 33 viruses, including 24 putative novel viral species. The most frequently detected viruses were phylogenetically related to members of the Bunyaviridae and Rhabdoviridae families, as well as the recently proposed Chuviridae . Our work expands our understanding of tick viromes and underscores the high viral diversity that is present in ticks. IMPORTANCE The incidence of tick-borne disease is increasing, driven by rapid geographical expansion of ticks and the discovery of new tick-associated pathogens. The examination of the tick microbiome is essential in order to understand the relationship between microbes and their tick hosts and to facilitate the identification of new tick-borne pathogens. Genomic analyses using unbiased high-throughput sequencing platforms have proven valuable for investigations of tick bacterial diversity, but the examination of tick viromes has historically not been well explored. By performing a comprehensive virome analysis of the three primary tick species associated with human disease in the United States, we gained substantial insight into tick virome diversity and can begin to assess a potential role of these viruses in the tick life cycle.

scholarly journals High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers

2021 ◽  
Vol 22 (17) ◽  
pp. 9202
Author(s):  
Alissa Drees ◽  
Markus Fischer
Keyword(s):  
High Throughput ◽  
High Performance ◽  
High Throughput Sequencing ◽  
Selection Process ◽  
Next Generation ◽  
Binding Assays ◽  
Ligand Interaction ◽  
Modified Dna ◽  
Sequencing Platforms ◽  
And Function

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. ‘High-throughput sequencing fluorescent ligand interaction profiling’ (HiTS–FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS–FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS–FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

scholarly journals Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

2013 ◽  
Vol 79 (8) ◽  
pp. 2519-2526 ◽  
Author(s):  
Nicholas A. Bokulich ◽  
David A. Mills
Keyword(s):  
High Throughput ◽  
Internal Transcribed Spacer ◽  
High Throughput Sequencing ◽  
Work Flow ◽  
Fungal Communities ◽  
Amplification Bias ◽  
Sequencing Technologies ◽  
Yeast Community ◽  
Primer Sets ◽  
Sequencing Platforms

ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

scholarly journals Access COI barcode efficiently using high throughput Single-End 400 bp sequencing

2018 ◽  
Author(s):  
Chentao Yang ◽  
Shangjin Tan ◽  
Guangliang Meng ◽  
David G. Bourne ◽  
Paul A. O’Brien ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rapid Development ◽  
Full Length ◽  
List Type ◽  
Sequencing Data ◽  
Test Plate ◽  
Function Modules ◽  
Sequencing Platforms

SummaryOver the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, constraints in barcoding costs led to unbalanced efforts which prevented accurate taxonomic identification for biodiversity studies.We present a high throughput sequencing approach based on the HIFI-SE pipeline which takes advantage of Single-End 400 bp (SE400) sequencing data generated by BGISEQ-500 to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons. HIFI-SE was written in Python and included four function modules of filter, assign, assembly and taxonomy.We applied the HIFI-SE to a test plate which contained 96 samples (30 corals, 64 insects and 2 blank controls) and delivered a total of 86 fully assembled HIFI COI barcodes. By comparing to their corresponding Sanger sequences (72 sequences available), it showed that most of the samples (98.61%, 71/72) were correctly and accurately assembled, including 46 samples that had a similarity of 100% and 25 of ca. 99%.Our approach can produce standard full-length barcodes cost efficiently, allowing DNA barcoding for global biomes which will advance DNA-based species identification for various ecosystems and improve quarantine biosecurity efforts.

scholarly journals In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing

2021 ◽  
Author(s):  
Fatimah Alhamlan ◽  
Dana Bakheet ◽  
Marie Bohol ◽  
Madain Alsanea ◽  
Basma Alahaideb ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Rna Extraction ◽  
Whole Genome ◽  
Diagnostic Laboratory ◽  
Spike Gene

Background: The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern. Methods: Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples. Results: Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical variants of concern results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). Moreover, the remaining samples were non-VOC that belonged to none of these variants and had 99.97% identity with the reference genome. Only four samples had poor sequence quality by Sanger sequencing owing to a low viral count (Ct value >38). Therefore, mutation calls were >98% accurate. Conclusions: Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.

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