Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation

2021 ◽  
Vol 61 (1_suppl) ◽  
pp. 92-95
Author(s):  
Stefania Turrina ◽  
Domenico De Leo
Keyword(s):  
Massively Parallel Sequencing ◽  
Forensic Genetics ◽  
Library Preparation ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Scale Down ◽  
Reaction Volumes ◽  
The Cost ◽  
Next Generation Sequencing Ngs ◽  
Dna Profiles

The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.

scholarly journals Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

2018 ◽  
Author(s):  
Madeline Y Mayday ◽  
Lillian M Khan ◽  
Eric D Chow ◽  
Matt S Zinter ◽  
Joseph L DeRisi
Keyword(s):  
Metagenomic Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Reaction Volumes ◽  
Sequencing Platforms ◽  
Time Required ◽  
The Cost ◽  
Sequencing Library Preparation ◽  
Full Volume ◽  
Generation Sequencing

AbstractPreparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

scholarly journals NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions

2020 ◽  
Vol 16 (12) ◽  
pp. e1008468
Author(s):  
Andrey Kechin ◽  
Viktoria Borobova ◽  
Ulyana Boyarskikh ◽  
Evgeniy Khrapov ◽  
Sergey Subbotin ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Primer Design ◽  
Nucleotide Polymorphisms ◽  
Chain Reactions ◽  
Targeted Next Generation Sequencing ◽  
Polymerase Chain Reactions ◽  
Coding Regions ◽  
Gene Coding ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.

scholarly journals Application of RAPD-PCR Markers for Identification and Genetic Analysis of American Elm (Ulmus americana L.) Selections

1995 ◽  
Vol 13 (4) ◽  
pp. 155-159
Author(s):  
Joseph C. Kamalay ◽  
David W. Carey
Keyword(s):  
Genetic Analysis ◽  
Cultured Cells ◽  
Easy Method ◽  
Ulmus Americana ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
American Elm ◽  
Rapd Pcr ◽  
Polymerase Chain ◽  
Dna Profiles

Abstract We used randomly amplified polymorphic DNA (RAPD) markers from DNA polymerase chain reactions to differentiate selected American elms. DNA profiles made from leaf DNAs of parental trees were identical to profiles produced by DNA from leaves, roots, vascular tissues, callus or suspension-cultured cells from the same trees or their ramets. In addition, several variations in the technique did not appear to alter the genotype-specific DNA profiles from a given elm selection. RAPD polymorphisms were inherited as dominant Mendelian factors in the progeny of a cross between two American elm selections. We conclude that the construction of RAPD DNA profiles is a reliable and easy method for selection identification, genetic analysis, and the assembly of linkage maps in American elms.

scholarly journals Investigation of Lethal Concurrent Outbreak of Chlamydiosis and Pigeon Circovirus in a Zoo

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1654
Author(s):  
Wei-Tao Chen ◽  
Chin-Ann Teng ◽  
Cheng-Hsin Shih ◽  
Wei-Hsiang Huang ◽  
Yi-Fan Jiang ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Chlamydia Psittaci ◽  
Bursa Of Fabricius ◽  
Chain Reactions ◽  
Renal Epithelium ◽  
Polymerase Chain Reactions ◽  
Transmission Electron ◽  
Intracytoplasmic Inclusion Bodies ◽  
Polymerase Chain ◽  
Streptopelia Orientalis

During the spring, an outbreak of sudden death involving 58 birds occurred in a zoo. Histopathological examinations revealed variable numbers of intracytoplasmic basophilic microorganisms in the macrophages, hepatocytes, and renal epithelium of most birds, along with occasional botryoid intracytoplasmic inclusion bodies within histiocytes in the bursa of Fabricius. Based on the results of histopathological examinations, immunohistochemical staining, transmission electron microscopy, and polymerase chain reactions, genotype B Chlamydia psittaci infection concurrent with pigeon circovirus (PiCV) was diagnosed. A retrospective survey, including two years before the outbreak and the outbreak year, of C. psittaci and PiCV infections of dead birds in the aviaries, revealed that the outbreak was an independent episode. The findings of this study indicate that concurrent infection with C. psittaci and PiCV might lead to lethal outbreaks of chlamydiosis, particularly Streptopelia orientalis. In addition, persistently monitoring both pathogens and identifying potential PiCV carriers or transmitters might also help prevent lethal disease outbreaks.

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