scholarly journals Performance comparison of four types of target enrichment baits for exome DNA sequencing

Hereditas ◽  
2021 ◽  
Vol 158 (1) ◽  
Author(s):  
Juan Zhou ◽  
Mancang Zhang ◽  
Xiaoqi Li ◽  
Zhuo Wang ◽  
Dun Pan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Deep Sequencing ◽  
Gc Content ◽  
Performance Comparison ◽  
Capture Efficiency ◽  
Next Generation ◽  
Double Stranded Rna ◽  
Target Capture ◽  
Binding Power ◽  
Generation Sequencing

Abstract Background Next-generation sequencing technology is developing rapidly and target capture sequencing has become an important technique. Several different platforms for library preparation and target capture with different bait types respectively are commercially available. Here we compare the performance of the four platforms with different bait types to find out their advantages and limitations. The purpose of this study is to help investigators and clinicians select the appropriate platform for their particular application and lay the foundation for the development of a better target capture platform for next-generation sequencing. Results We formulate capture efficiency as a novel parameter that can be used to better evaluations of specificity and coverage depth among the different capture platforms. Target coverage, capture efficiency, GC bias, AT Dropout, sensitivity in single nucleotide polymorphisms, small insertions and deletions detection, and the feature of each platform were compared for low input samples. In general, all platforms perform well and small differences among them are revealed. In our results, RNA baits have stronger binding power than DNA baits, and with ultra deep sequencing, double stranded RNA baits perform better than single stranded RNA baits in all aspects. DNA baits got better performance in the region with high GC content and RNA baits got lower AT dropout suggesting that the binding power is different between DNA and RNA baits to genome regions with different characteristics. Conclusions The platforms with double stranded RNA baits have the most balanced capture performance. Our results show the key differences in performance among the four updated platforms with four different bait types. The better performance of double stranded RNA bait with ultra deep sequencing suggests that it may improve the sensitivity of ultra low frequent mutation detection. In addition, we further propose that the mixed baits of double stranded RNA and single stranded DNA may improve target capture performance.

scholarly journals Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance

2018 ◽  
Vol 62 (4) ◽  
pp. e02474-17 ◽  
Author(s):  
Eldin Talundzic ◽  
Shashidhar Ravishankar ◽  
Julia Kelley ◽  
Dhruviben Patel ◽  
Mateusz Plucinski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Deep Sequencing ◽  
Artemisinin Resistance ◽  
The United States ◽  
Imported Malaria ◽  
Chloroquine Resistance ◽  
Next Generation ◽  
Content Type ◽  
Generation Sequencing

ABSTRACT The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

scholarly journals Performance Comparison of Different Analytic Methods in Proficiency Testing for Mutations in the BRAF, EGFR, and KRAS Genes: A Study of the College of American Pathologists Molecular Oncology Committee

2019 ◽  
Vol 143 (10) ◽  
pp. 1203-1211 ◽  
Author(s):  
Joel T. Moncur ◽  
Angela N. Bartley ◽  
Julia A. Bridge ◽  
Suzanne Kamel-Reid ◽  
Alexander J. Lazar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
Critical Issue ◽  
Performance Comparison ◽  
Test Method ◽  
Next Generation ◽  
Molecular Oncology ◽  
Assay Methods ◽  
Commercial Kits ◽  
Generation Sequencing

Context.— The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories (“home brews”), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee. Objective.— To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS. Design.— There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices. Results.— Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing. Conclusions.— These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

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 Target capture next‐generation sequencing in non‐inversion haemophilia: an alternative approach

2020 ◽  
Vol 189 (4) ◽  
Author(s):  
Qiang Li ◽  
Juanjuan Chen ◽  
Sheng Lin ◽  
Limin Huang ◽  
Xu Yang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Target Capture ◽  
Alternative Approach ◽  
Generation Sequencing
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