scholarly journals Author Correction: Ultrasensitive amplicon barcoding for next-generation sequencing facilitating sequence error and amplification-bias correction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ibrahim Ahmed ◽  
Felicia A. Tucci ◽  
Aure Aflalo ◽  
Kenneth G. C. Smith ◽  
Rachael J. M. Bashford-Rogers
Keyword(s):  
Next Generation Sequencing ◽  
Bias Correction ◽  
Next Generation ◽  
Amplification Bias ◽  
Sequence Error ◽  
Generation Sequencing

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

scholarly journals Ultrasensitive amplicon barcoding for next-generation sequencing facilitating sequence error and amplification-bias correction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ibrahim Ahmed ◽  
Felicia A. Tucci ◽  
Aure Aflalo ◽  
Kenneth G. C. Smith ◽  
Rachael J. M. Bashford-Rogers
Keyword(s):  
Next Generation Sequencing ◽  
Bias Correction ◽  
Tumor Heterogeneity ◽  
Pcr Amplification ◽  
Genetic Studies ◽  
Immune Receptor ◽  
Amplification Bias ◽  
Sequencing Errors ◽  
Sequence Error ◽  
Generation Sequencing

Abstract The ability to accurately characterize DNA variant proportions using PCR amplification is key to many genetic studies, including studying tumor heterogeneity, 16S microbiome, viral and immune receptor sequencing. We develop a novel generalizable ultrasensitive amplicon barcoding approach that significantly reduces the inflation/deflation of DNA variant proportions due to PCR amplification biases and sequencing errors. This method was applied to immune receptor sequencing, where it significantly improves the quality and estimation of diversity of the resulting library.

Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms

2016 ◽  
Vol 69 (9) ◽  
pp. 801-804 ◽  
Author(s):  
Benedict Yan ◽  
Yongli Hu ◽  
Christopher Ng ◽  
Kenneth H K Ban ◽  
Tin Wee Tan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gc Content ◽  
Read Depth ◽  
Next Generation ◽  
Diagnostic Assays ◽  
Amplification Bias ◽  
Lower Performance ◽  
Myeloid Neoplasms ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AimsPCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA.MethodsWe performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow.ResultsWe identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples.ConclusionsThe identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.

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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