RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing

ABSTRACTHere, we present GAVIN, a new method that delivers accurate classification of variants for next-generation sequencing molecular diagnostics. It is based on gene-specific calibrations of allele frequencies (from the ExAC database), effect impact (using SnpEff) and estimated deleteriousness (CADD scores) for >3,000 genes. In a benchmark on 18 clinical gene sets, we achieved a sensitivity of 91.6%, with a specificity of 78.2%. This accuracy was unmatched by 12 other tools we tested. We provide GAVIN as an online MOLGENIS service to annotate VCF files, and as open source executable for use in bioinformatic pipelines. It can be found athttp://molgenis.org/gavin.

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Fragmentation Through Polymerization (FTP): A New Method to Fragment DNA for Next-Generation Sequencing

10.1101/505966 ◽

2018 ◽

Author(s):

Konstantin B. Ignatov ◽

Konstantin A. Blagodatskikh ◽

Dmitry S. Shcherbo ◽

Tatiana Kramarova ◽

Yulia A. Monakhova ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acids ◽

Low Cost ◽

New Method ◽

Enzymatic Method ◽

Next Generation ◽

Double Stranded Dna ◽

Dna Ends ◽

Direct Use ◽

Generation Sequencing

Fragmentation of DNA is the first and very important step in preparing nucleic acids for NGS. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is simple, robust and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for the direct use in NGS library construction, and allows to eliminate the need of an additional step of reparation of DNA ends.

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A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping

Microorganisms ◽

10.3390/microorganisms8091391 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1391

Author(s):

Flavia Hebeler-Barbosa ◽

Ivan Rodrigo Wolf ◽

Guilherme Targino Valente ◽

Francisco Campello do Amaral Mello ◽

Elisabeth Lampe ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Sanger Sequencing ◽

Clinical Specimen ◽

New Method ◽

Next Generation ◽

B Virus ◽

Reverse Hybridization ◽

Generation Sequencing

Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A–H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.

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