scholarly journals Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 929 ◽  
Author(s):  
Federica Alessandrini ◽  
Sara Caucci ◽  
Valerio Onofri ◽  
Filomena Melchionda ◽  
Adriano Tagliabracci ◽  
...  
Keyword(s):  
High Throughput ◽  
Reverse Transcription ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Relative Depth ◽  
High Throughput Analysis ◽  
Parallel Sequencing ◽  
Reverse Transcription Reaction

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer’s instructions.

scholarly journals Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

2021 ◽  
Author(s):  
Stella C. Yuan ◽  
Eric Malekos ◽  
Melissa T. R. Hawkins
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Museum Specimens ◽  
Museum Specimen ◽  
Genotyping Errors ◽  
Allelic Dropout ◽  
Parallel Sequencing ◽  
Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

scholarly journals High throughput whole mitochondrial genome sequencing by two platforms of massively parallel sequencing

BMC Genomics ◽  
2014 ◽  
Vol 15 (Suppl 2) ◽  
pp. P7 ◽  
Author(s):  
Seung Seo ◽  
Xiangpei Zeng ◽  
Mourad Assidi ◽  
Bobby LaRue ◽  
Jonathan King ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Genome Sequencing ◽  
High Throughput ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing

Abstract LB-122: High-throughput tumor genomic profiling by massively parallel sequencing

2010 ◽  
Author(s):  
Nikhil Wagle ◽  
Matt Davis ◽  
Michael F. Berger ◽  
Brendan Blumenstiel ◽  
Matthew Defelice ◽  
...  
Keyword(s):  
High Throughput ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Genomic Profiling ◽  
Parallel Sequencing

scholarly journals Massively parallel sequencing of 25 autosomal STRs including SE33 in four population groups for forensic applications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ye-Lim Kwon ◽  
Bo Min Kim ◽  
Eun Young Lee ◽  
Kyoung-Jin Shin
Keyword(s):  
Large Scale ◽  
Tandem Repeats ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Population Data ◽  
Massively Parallel ◽  
Autosomal Strs ◽  
Data Set ◽  
Parallel Sequencing ◽  
Sequence Variations

AbstractThe introduction of massively parallel sequencing (MPS) in forensic investigation enables sequence-based large-scale multiplexing beyond size-based analysis using capillary electrophoresis (CE). For the practical application of MPS to forensic casework, many population studies have provided sequence data for autosomal short tandem repeats (STRs). However, SE33, a highly polymorphic STR marker, has little sequence-based data because of difficulties in analysis. In this study, 25 autosomal STRs were analyzed, including SE33, using an in-house MPS panel for 350 samples from four populations (African–American, Caucasian, Hispanic, and Korean). The barcoded MPS library was generated using a two-step PCR method and sequenced using a MiSeq System. As a result, 99.88% genotype concordance was obtained between length- and sequence-based analyses. In SE33, the most discordances (eight samples, 0.08%) were observed because of the 4 bp deletion between the CE and MPS primer binding sites. Compared with the length-based CE method, the number of alleles increased from 332 to 725 (2.18-fold) for 25 autosomal STRs in the sequence-based MPS method. Notably, additional 129 unique alleles, a 4.15-fold increase, were detected in SE33 by identifying sequence variations. This population data set provides sequence variations and sequence-based allele frequencies for 25 autosomal STRs.

scholarly journals Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19

2020 ◽  
Author(s):  
Mayu Ikeda ◽  
Kazuo Imai ◽  
Sakiko Tabata ◽  
Kazuyasu Miyoshi ◽  
Nami Murahara ◽  
...  
Keyword(s):  
High Throughput ◽  
Reverse Transcription ◽  
Symptom Onset ◽  
Diagnostic Tests ◽  
Clinical Performance ◽  
Viral Rna ◽  
Molecular Diagnostic ◽  
Clinical Use ◽  
Antigen Test ◽  
Asymptomatic Patients

AbstractBackgroundThe clinical performance of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva.MethodsSaliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) laboratory-developed tes (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse-transcription loop mediated isothermal amplification (RT-LAMP). Viral antigen was detected by a rapid antigen immunochromatographic assay.ResultsOf the 103 samples, viral RNA was detected in 50.5–81.6% of the specimens by molecular diagnostic tests and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at a significantly higher percentage (65.6–93.4%) in specimens collected within 9 d of symptom onset compared to that of specimens collected after at least 10 d of symptom onset (22.2–66.7%) and that of asymptomatic patients (40.0–66.7%). Viral RNA was more frequently detected in saliva from males than females.ConclusionsSelf-collected saliva is an alternative specimen diagnosing COVID-19. LDT RT-qPCR, cobas SARS-CoV-2 high-throughput system, direct RT-qPCR except for one commercial kit, and RT-LAMP showed sufficient sensitivity in clinical use to be selectively used according to clinical settings and facilities. The rapid antigen test alone is not recommended for initial COVID-19 diagnosis because of its low sensitivity.Key pointsSix molecular diagnostic tests showed equivalent and sufficient sensitivity in clinical use in diagnosing COVID-19 in self-collected saliva samples. However, a rapid SARS-CoV-2 antigen test alone is not recommended for use without further study.

scholarly journals PhenoMIP: High-Throughput Phenotyping of Diverse Caenorhabditis elegans Populations via Molecular Inversion Probes

2020 ◽  
Vol 10 (11) ◽  
pp. 3977-3990
Author(s):  
Calvin Mok ◽  
Gabriella Belmarez ◽  
Mark L. Edgley ◽  
Donald G. Moerman ◽  
Robert H. Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Massively Parallel Sequencing ◽  
Food Sources ◽  
Relative Fitness ◽  
Model Organisms ◽  
Single Nucleotide Variants ◽  
High Throughput Analysis ◽  
Link Type ◽  
Mutant Strains

Whether generated within a lab setting or isolated from the wild, variant alleles continue to be an important resource for decoding gene function in model organisms such as Caenorhabditis elegans. With advances in massively parallel sequencing, multiple whole-genome sequenced (WGS) strain collections are now available to the research community. The Million Mutation Project (MMP) for instance, analyzed 2007 N2-derived, mutagenized strains. Individually, each strain averages ∼400 single nucleotide variants amounting to ∼80 protein-coding variants. The effects of these variants, however, remain largely uncharacterized and querying the breadth of these strains for phenotypic changes requires a method amenable to rapid and sensitive high-throughput analysis. Here we present a pooled competitive fitness approach to quantitatively phenotype subpopulations of sequenced collections via molecular inversion probes (PhenoMIP). We phenotyped the relative fitness of 217 mutant strains on multiple food sources and classified these into five categories. We also demonstrate on a subset of these strains, that their fitness defects can be genetically mapped. Overall, our results suggest that approximately 80% of MMP mutant strains may have a decreased fitness relative to the lab reference, N2. The costs of generating this form of analysis through WGS methods would be prohibitive while PhenoMIP analysis in this manner is accomplished at less than one-tenth of projected WGS costs. We propose methods for applying PhenoMIP to a broad range of population selection experiments in a cost-efficient manner that would be useful to the community at large.

Sign in / Sign up

Export Citation Format

Share Document