Filling the gap of short-read next generation sequencing in PGD by long-read approach

2018 ◽  
Author(s):  
Dona Ngar Yin Ho
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Short Read ◽  
Long Read ◽  
Generation Sequencing

scholarly journals Potential of whole-genome sequencing-based pharmacogenetic profiling

2021 ◽  
Author(s):  
Sylvan Manuel Caspar ◽  
Timo Schneider ◽  
Patricia Stoll ◽  
Janine Meienberg ◽  
Gabor Matyas
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Next Generation ◽  
Short Read ◽  
Long Read ◽  
Improving Patient Care ◽  
Gene Diagnostics ◽  
Generation Sequencing

Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

PERHAPS: Paired-End short Reads-based HAPlotyping from next-generation Sequencing data

2020 ◽  
Author(s):  
Jie Huang ◽  
Stefano Pallotti ◽  
Qianling Zhou ◽  
Marcus Kleber ◽  
Xiaomeng Xin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Snp Array ◽  
Simple Approach ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Short Read ◽  
Array Data ◽  
Short Reads ◽  
Generation Sequencing

Abstract The identification of rare haplotypes may greatly expand our knowledge in the genetic architecture of both complex and monogenic traits. To this aim, we developed PERHAPS (Paired-End short Reads-based HAPlotyping from next-generation Sequencing data), a new and simple approach to directly call haplotypes from short-read, paired-end Next Generation Sequencing (NGS) data. To benchmark this method, we considered the APOE classic polymorphism (*1/*2/*3/*4), since it represents one of the best examples of functional polymorphism arising from the haplotype combination of two Single Nucleotide Polymorphisms (SNPs). We leveraged the big Whole Exome Sequencing (WES) and SNP-array data obtained from the multi-ethnic UK BioBank (UKBB, N=48,855). By applying PERHAPS, based on piecing together the paired-end reads according to their FASTQ-labels, we extracted the haplotype data, along with their frequencies and the individual diplotype. Concordance rates between WES directly called diplotypes and the ones generated through statistical pre-phasing and imputation of SNP-array data are extremely high (>99%), either when stratifying the sample by SNP-array genotyping batch or self-reported ethnic group. Hardy-Weinberg Equilibrium tests and the comparison of obtained haplotype frequencies with the ones available from the 1000 Genome Project further supported the reliability of PERHAPS. Notably, we were able to determine the existence of the rare APOE*1 haplotype in two unrelated African subjects from UKBB, supporting its presence at appreciable frequency (approximatively 0.5%) in the African Yoruba population. Despite acknowledging some technical shortcomings, PERHAPS represents a novel and simple approach that will partly overcome the limitations in direct haplotype calling from short read-based sequencing.

scholarly journals Methods for analyzing next-generation sequencing data VII. long-read assembly

2016 ◽  
Vol 27 (2) ◽  
pp. 101-110
Author(s):  
Yasuhiro Tanizawa ◽  
Eli Kaminuma ◽  
Yasukazu Nakamura ◽  
Masanori Tohno ◽  
Ken Osaki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Long Read ◽  
Generation Sequencing

scholarly journals MOSAIK: A Hash-Based Algorithm for Accurate Next-Generation Sequencing Short-Read Mapping

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e90581 ◽  
Author(s):  
Wan-Ping Lee ◽  
Michael P. Stromberg ◽  
Alistair Ward ◽  
Chip Stewart ◽  
Erik P. Garrison ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Read Mapping ◽  
Short Read ◽  
Short Read Mapping ◽  
Generation Sequencing

scholarly journals Next-generation sequencing of double stranded RNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO

2019 ◽  
Author(s):  
Alexander H. Wilcox ◽  
Eric Delwart ◽  
Samuel L. Díaz Muñoz
Keyword(s):  
Next Generation Sequencing ◽  
Limit Of Detection ◽  
Genetic Material ◽  
Dsrna Virus ◽  
Next Generation ◽  
Short Read ◽  
Double Stranded Rna ◽  
Ncbi Short Read Archive ◽  
Dmso Treatment ◽  
Generation Sequencing

AbstractDouble stranded RNA (dsRNA) is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. Dimethyl sulfoxide (DMSO) has been used to denature dsRNA prior to the reverse transcription stage to improve RT-PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve sequencing yield of a dsRNA virus (Φ6) in a short-read next generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a single-stranded RNA viral genome (Influenza A/California/07/2009). We suggest that up to 50% DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.Data SummarySequence data was deposited in the NCBI Short Read Archive (accession numbers: PRJNA527100, PRJNA527101, PRJNA527098). Data and code for analysis is available on GitHub (https://github.com/awilcox83/dsRNA-sequencing/, doi:10.5281/zenodo.1453423). Protocol for dsRNA sequencing is posted on protocols.io (doi:10.17504/protocols.io.ugnetve).

scholarly journals Exogene: A performant workflow for detecting viral integrations from paired-end next-generation sequencing data

2021 ◽  
Author(s):  
Jean-Pierre Kocher ◽  
Zachary Stephens ◽  
Daniel O'Brien ◽  
Mrunal Dehankar ◽  
Lewis Roberts ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Data ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Long Read ◽  
Breakpoint Detection ◽  
Targeted Capture ◽  
Genome Heterogeneity ◽  
Generation Sequencing

The integration of viruses into the human genome is known to be associated with tumorigenesis in many cancers, but the accurate detection of integration breakpoints from short read sequencing data is made difficult by human-viral homologies, viral genome heterogeneity, coverage limitations, and other factors. To address this, we present Exogene, a sensitive and efficient workflow for detecting viral integrations from paired-end next generation sequencing data. Exogene's read filtering and breakpoint detection strategies yield integration coordinates that are highly concordant with those found in long read validation sets. We demonstrate this concordance across 6 TCGA Hepatocellular carcinoma (HCC) tumor samples, identifying integrations of hepatitis B virus that are validated by long reads. Additionally, we applied Exogene to targeted capture data from 426 previously studied HCC samples, achieving 98.9% concordance with existing methods and identifying 238 high-confidence integrations that were not previously reported. Exogene is applicable to multiple types of paired-end sequence data, including genome, exome, RNA-Seq or targeted capture.

scholarly journals Chromosome and Plasmids of the Tick-Borne Relapsing Fever Agent Borrelia hermsii

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Alan G. Barbour
Keyword(s):  
Next Generation Sequencing ◽  
Linear Plasmids ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Linear Chromosome ◽  
Short Read ◽  
Zoonotic Pathogen ◽  
Long Read ◽  
Complete Sequences ◽  
Generation Sequencing

The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome.

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