scholarly journals Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms

2017 ◽  
Author(s):  
Maura Costello ◽  
Mark Fleharty ◽  
Justin Abreu ◽  
Yossi Farjoun ◽  
Steven Ferriera ◽  
...  
Keyword(s):  
Best Practices ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Preparation Methods ◽  
Multiple Sample ◽  
Cluster Generation ◽  
Sequencing Platforms ◽  
Insight Into ◽  
Swap Rates

ABSTRACTHere, we present an in-depth characterization of the index swapping mechanism on Illumina instruments that employ the ExAmp chemistry for cluster generation (HiSeqX, HiSeq4000, and NovaSeq). We discuss best practices for eliminating the effects of index swapping on data integrity by utilizing unique dual indexing for complete filtering of index swapped reads. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different methods can have vastly different swap rates, and show that even non-ExAmp chemistry instruments display trace levels of index swapping. Finally, using computational methods we provide a greater insight into the mechanism of index swapping.

scholarly journals Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

2018 ◽  
Author(s):  
Qiaoling Li ◽  
Xia Zhao ◽  
Wenwei Zhang ◽  
Lin Wang ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Rolling Circle Replication ◽  
Linear Amplification ◽  
Preparation Methods ◽  
Rolling Circle ◽  
Quality Issue ◽  
Error Accumulation ◽  
Sequencing Platforms ◽  
Multiplex Sequencing

AbstractBackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.ResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI’s sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.ConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

scholarly journals Comparison of Normalization Methods for Construction of Large, Multiplex Amplicon Pools for Next-Generation Sequencing

2010 ◽  
Vol 76 (12) ◽  
pp. 3863-3868 ◽  
Author(s):  
J. Kirk Harris ◽  
Jason W. Sahl ◽  
Todd A. Castoe ◽  
Brandie D. Wagner ◽  
David D. Pollock ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Cost Savings ◽  
Massively Parallel ◽  
Next Generation ◽  
Normalization Methods ◽  
The Cost ◽  
Generation Sequencing

ABSTRACT Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.

Abstract 1139: Complete characterization of prostate cancer genomes by massively parallel sequencing

2010 ◽  
Author(s):  
Michael F. Berger ◽  
Michael S. Lawrence ◽  
Kristian Cibulskis ◽  
Dorothee Pflueger ◽  
Francesca Demichelis ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Massively Parallel Sequencing ◽  
Complete Characterization ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Cancer Genomes

scholarly journals Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e104566 ◽  
Author(s):  
Carina Heydt ◽  
Jana Fassunke ◽  
Helen Künstlinger ◽  
Michaela Angelika Ihle ◽  
Katharina König ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Massively Parallel Sequencing ◽  
Ffpe Sample ◽  
Massively Parallel ◽  
Preparation Methods ◽  
Routine Diagnostics ◽  
Parallel Sequencing ◽  
Sample Preparation Methods

scholarly journals Molecular Characterization of Human T-Cell Lymphotropic Virus Type 1 Full and Partial Genomes by Illumina Massively Parallel Sequencing Technology

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e93374 ◽  
Author(s):  
Rodrigo Pessôa ◽  
Jaqueline Tomoko Watanabe ◽  
Youko Nukui ◽  
Juliana Pereira ◽  
Jorge Kasseb ◽  
...  
Keyword(s):  
T Cell ◽  
Molecular Characterization ◽  
Virus Type ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Sequencing Technology ◽  
Parallel Sequencing ◽  
Human T Cell

scholarly journals Visualization tools for human structural variations identified by whole-genome sequencing

2019 ◽  
Vol 65 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Toshiyuki T. Yokoyama ◽  
Masahiro Kasahara
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Visualization Tool ◽  
Structural Variations ◽  
Long Read ◽  
Comprehensive Survey ◽  
Visualization Tools ◽  
Sequencing Platforms ◽  
Near Future ◽  
Individual Human

Abstract Visualizing structural variations (SVs) is a critical step for finding associations between SVs and human traits or diseases. Given that there are many sequencing platforms used for SV identification and given that how best to visualize SVs together with other data, such as read alignments and annotations, depends on research goals, there are dozens of SV visualization tools designed for different research goals and sequencing platforms. Here, we provide a comprehensive survey of over 30 SV visualization tools to help users choose which tools to use. This review targets users who wish to visualize a set of SVs identified from the massively parallel sequencing reads of an individual human genome. We first categorize the ways in which SV visualization tools display SVs into ten major categories, which we denote as view modules. View modules allow readers to understand the features of each SV visualization tool quickly. Next, we introduce the features of individual SV visualization tools from several aspects, including whether SV views are integrated with annotations, whether long-read alignment is displayed, whether underlying data structures are graph-based, the type of SVs shown, whether auditing is possible, whether bird’s eye view is available, sequencing platforms, and the number of samples. We hope that this review will serve as a guide for readers on the currently available SV visualization tools and lead to the development of new SV visualization tools in the near future.

scholarly journals Robustness of Massively Parallel Sequencing Platforms

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138259 ◽  
Author(s):  
Pınar Kavak ◽  
Bayram Yüksel ◽  
Soner Aksu ◽  
M. Oguzhan Kulekci ◽  
Tunga Güngör ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Sequencing Platforms

scholarly journals Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Maura Costello ◽  
Mark Fleharty ◽  
Justin Abreu ◽  
Yossi Farjoun ◽  
Steven Ferriera ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Sequencing Platforms
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