Fusion sequencing via terminator-assisted synthesis (FTAS-seq) identifies TMPRSS2 fusion partners in prostate cancer.

Genetic rearrangements that fuse an androgen-regulated promoter area with a protein-coding portion of an originally androgen-unaffected gene are frequent in prostate cancer, with the fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2-ERG fusion) being the most prevalent. Conventional hybridization- or amplification-based methods can test for the presence of expected gene fusions, but the exploratory analysis of currently unknown fusion partners is often cost-prohibitive. Here, we developed an innovative next-generation sequencing (NGS)-based approach for gene fusion analysis termed fusion sequencing via terminator-assisted synthesis (FTAS-seq).

Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2',3'-dideoxynucleotides.

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications.

Sensitive and accurate analysis of gene expression signatures enabled by oligonucleotide-labelled cDNA.

High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3' termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) - a novel RNA-seq library preparation method directed towards mRNA 3' termini.

High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts.

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1-V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene.

MuA-based Molecular Indexing for Rare Mutation Detection by Next-Generation Sequencing.

Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases.

Enzymatic Synthesis of Chimeric DNA Oligonucleotides by Transcription with dTTP, dCTP, dATP, and 2'-Fluoro Modified dGTP.

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination.

Author Correction: Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

The original version of this Article contained errors in the units of concentration of three reagents listed in the Methods. These errors have all been corrected in both the PDF and HTML versions of the Article.

UbaLAI is a monomeric Type IIE restriction enzyme.

Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position).

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability.

Draft Genome Sequence of the Cyanobacterium Aphanizomenon flos-aquae Strain 2012/KM1/D3, Isolated from the Curonian Lagoon (Baltic Sea).

We report here the de novo genome assembly of a cyanobacterium, Aphanizomenon flos-aquae strain 2012/KM1/D3, a harmful bloom-forming species in temperate aquatic ecosystems. The genome is 5.7 Mb with a G+C content of 38.

A new prototype IIS/IIC/IIG endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus, recognising 5'-TARCCA(N11/9)-3' sequences.

The Thermus sp. family of IIS/IIG/IIC enzymes includes the thermostable, bifunctional, fused restriction endonuclease (REase)-methyltransferases (MTase): TaqII, Tth111II/TthHB27I, TspGWI, TspDTI and TsoI. The enzymes are large proteins (approximately 120kDa), their enzymatic activities are affected by S-adenosylmethionine (SAM), they recognise similar asymmetric cognate sites and cleave at a distance of 11/9 nucleotides (nt).

Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus.

Background: In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively.

Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency.

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success.

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities.

Background: We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.

Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well.

Bioinformatic and partial functional analysis of pEspA and pEspB, two plasmids from Exiguobacterium arabatum sp. nov. RFL1109.

The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined.

Synthesis of four colors fluorescently labelled 3'-O-blocked nucleotides with fluoride cleavable blocking group and linker for array based Sequencing-by-Synthesis applications.

Reversible terminators having a fluoride cleavable 3'-O-blocking group are presented. Each nucleotide triphosphate is labelled by a fluorescent dye cleavable by the same reagent. We present here their synthesis, cleavage experiments and polymerase incorporation tests for a possible use in a process of Sequencing-by-Synthesis.

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains.

Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity.

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease.

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database.

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains.

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA.

Random gene dissection: a tool for the investigation of protein structural organization.

To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI.

MnlI--The member of H-N-H subtype of Type IIS restriction endonucleases.

The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 downward arrow and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease.

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity.

Esp1396I restriction-modification system: structural organization and mode of regulation.

Esp1396I restriction-modification (RM) system recognizes an interrupted palindromic DNA sequence 5'-CCA(N)(5)TGG-3'. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp.