scholarly journals Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next‑generation sequencing

2019 ◽  
Author(s):  
Satoshi Fujii ◽  
Takayuki Yoshino ◽  
Kentaro Yamazaki ◽  
Kei Muro ◽  
Kensei Yamaguchi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
High Quality ◽  
Factors Affecting ◽  
Tissue Sections ◽  
Generation Sequencing

scholarly journals Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue

2019 ◽  
Vol 2 (2) ◽  
pp. 54 ◽  
Author(s):  
Md Masud Rana ◽  
Murat Aycan ◽  
Takeshi Takamatsu ◽  
Kentaro Kaneko ◽  
Toshiaki Mitsui ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Leaf Tissue ◽  
Isoamyl Alcohol ◽  
Phenol Removal ◽  
Library Preparation ◽  
Next Generation ◽  
High Quality ◽  
Fresh Leaf ◽  
Generation Sequencing

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

scholarly journals Targeted enrichment of genomic DNA regions for next-generation sequencing

2011 ◽  
Vol 10 (6) ◽  
pp. 374-386 ◽  
Author(s):  
F. Mertes ◽  
A. ElSharawy ◽  
S. Sauer ◽  
J. M. L. M. van Helvoort ◽  
P. J. van der Zaag ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
Generation Sequencing ◽  
Targeted Enrichment

Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing

2018 ◽  
Vol 3 (2) ◽  
pp. 178-184 ◽  
Author(s):  
M Rabie Al-Turkmani ◽  
Kelley N Godwin ◽  
Jason D Peterson ◽  
Gregory J Tsongalis
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Ffpe Tissue ◽  
Braf V600e ◽  
Automated System ◽  
Next Generation ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Single Use ◽  
Generation Sequencing

AbstractBackgroundMolecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples.MethodsForty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate.ResultsThe Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h.ConclusionThe Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

Factors Affecting Migration to GRCh38 in Laboratories Performing Clinical Next-Generation Sequencing

2021 ◽  
Vol 23 (5) ◽  
pp. 651-657
Author(s):  
Lisa A. Lansdon ◽  
Maxime Cadieux-Dion ◽  
Byunggil Yoo ◽  
Neil Miller ◽  
Ana S.A. Cohen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Factors Affecting ◽  
Generation Sequencing

scholarly journals Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

Genes ◽  
2014 ◽  
Vol 5 (4) ◽  
pp. 1115-1131 ◽  
Author(s):  
Yukio Kurihara ◽  
Yuko Makita ◽  
Mika Kawashima ◽  
Hidefumi Hamasaki ◽  
Yoshiharu Yamamoto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Dna Fragments ◽  
Next Generation ◽  
Regulatory Potential ◽  
Generation Sequencing

scholarly journals Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

2020 ◽  
Vol 8 (1) ◽  
pp. e000299
Author(s):  
Ping Zhang ◽  
Devika Ganesamoorthy ◽  
Son Hoang Nguyen ◽  
Raymond Au ◽  
Lachlan J Coin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Cost Effective ◽  
Inverse Pcr ◽  
Nanopore Sequencing ◽  
Next Generation ◽  
Short Read ◽  
Vector Integration ◽  
Integration Sites ◽  
Generation Sequencing

BackgroundAnalysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs.MethodsWe used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters,NcoIandBspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters.ResultsBoth inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3′long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing.ConclusionOur study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

scholarly journals Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1691
Author(s):  
Muscarella ◽  
Fabrizio ◽  
De Bonis ◽  
Mancini ◽  
Balsamo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Scale Up ◽  
Cancer Diagnostics ◽  
Sequencing Analysis ◽  
Library Preparation ◽  
Next Generation ◽  
High Quality ◽  
High Coverage ◽  
Dna Recovery ◽  
Generation Sequencing

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

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