scholarly journals Logarithmic molecular sampling for next-generation sequencing

2018 ◽  
Author(s):  
Caroline Horn ◽  
Julia Salzman
Keyword(s):  
Next Generation Sequencing ◽  
Fundamental Problem ◽  
Next Generation ◽  
Signal To Noise ◽  
Sampling Depth ◽  
Entropy Encoding ◽  
New Generation ◽  
Generation Sequencing ◽  
Conventional Sequencing ◽  
Proof Of Principle

AbstractNext-generation sequencing enables measurement of chemical and biological signals at high throughput and falling cost. Conventional sequencing requires increasing sampling depth to improve signal to noise discrimination, a costly procedure that is also impossible when biological material is limiting. We introduce a new general sampling theory, Molecular Entropy encodinG (MEG), which uses biophysical principles to functionally encode molecular abundance before sampling. SeQUential DepletIon and enriCHment (SQUICH) is a specific example of MEG that, in theory and simulation, enables sampling at a logarithmic or better rate to achieve the same precision as attained with conventional sequencing. In proof-of-principle experiments, SQUICH reduces sequencing depth by a factor of 10. MEG is a general solution to a fundamental problem in molecular sampling and enables a new generation of efficient, precise molecular measurement at logarithmic or better sampling depth.

scholarly journals A Targeted “Next-Generation” Sequencing-Informatic Approach to Define Genetic Diversity in Theileria orientalis Populations within Individual Cattle: Proof-of-Principle

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 448
Author(s):  
Anson V. Koehler ◽  
Abdul Jabbar ◽  
Ross S. Hall ◽  
Robin B. Gasser
Keyword(s):  
Genetic Variation ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Asia Pacific ◽  
Next Generation ◽  
Gene Markers ◽  
Theileria Orientalis ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing ◽  
Proof Of Principle

Oriental theileriosis is an economically important tickborne disease of bovines, caused by some members of the Theileria orientalis complex. Currently, 11 distinct operational taxonomic units (OTUs), or genotypes, are recognized based on their major piroplasm surface protein (MPSP) gene sequences. Two of these genotypes (i.e., chitose and ikeda) are recognized as pathogenic in cattle, causing significant disease in countries of the Asia-Pacific region. However, the true extent of genetic variation and associated virulence/pathogenicity within this complex is unknown. Here, we undertook a proof-of-principle study of a small panel of genomic DNAs (n = 13) from blood samples originating from individual cattle known to harbor T. orientalis, in order to assess the performance of a targeted “next-generation” sequencing-informatic approach to identify genotypes. Five genotypes (chitose, ikeda, buffeli, type 4, and type 5) were defined; multiple genotypes were found within individual samples, with dominant and minor sequence types representing most genotypes. This study indicates that this sequencing-informatic workflow could be useful to assess the nature and extent of genetic variation within and among populations of T. orientalis on a large scale, and to potentially employ panels of distinct gene markers for expanded molecular epidemiological investigations of socioeconomically important protistan pathogens more generally.

scholarly journals Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

2015 ◽  
Vol 89 (16) ◽  
pp. 8540-8555 ◽  
Author(s):  
Shuntai Zhou ◽  
Corbin Jones ◽  
Piotr Mieczkowski ◽  
Ronald Swanstrom
Keyword(s):  
Next Generation Sequencing ◽  
Error Rate ◽  
Consensus Sequence ◽  
Next Generation ◽  
Sampling Depth ◽  
Data Set ◽  
Sequencing Errors ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1

ABSTRACTValidating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS.IMPORTANCEAlthough next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.

Labordiagnostik bei gastrointestinalen Tumoren

2020 ◽  
Vol 11 (05) ◽  
pp. 232-238
Author(s):  
Marcus Kleber
Keyword(s):  
Next Generation Sequencing ◽  
Kolorektales Karzinom ◽  
Next Generation ◽  
Generation Sequencing

ZUSAMMENFASSUNGDas kolorektale Karzinom (KRK) ist einer der häufigsten malignen Tumoren in Deutschland. Einer frühzeitigen Diagnostik kommt große Bedeutung zu. Goldstandard ist hier die Koloskopie. Die aktuelle S3-Leitlinie Kolorektales Karzinom empfiehlt zum KRK-Screening den fäkalen okkulten Bluttest. Für das Monitoring von Patienten vor und nach Tumorresektion werden die Messung des Carcinoembryonalen Antigens (CEA) und der Mikrosatellitenstabilität empfohlen. Für die Auswahl der korrekten Chemotherapie scheint derzeit eine Überprüfung des Mutationsstatus, mindestens des KRAS-Gens und des BRAF-Gens, sinnvoll zu sein. Eine Reihe an neuartigen Tumormarkern befindet sich momentan in der Entwicklung, hat jedoch noch nicht die Reife für eine mögliche Anwendung in der Routinediagnostik erreicht. Den schnellsten Weg in die breite Anwendung können Next-Generation-Sequencing-basierte genetische Tests finden.

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