Massively parallel DNA target capture using Long Adapter Single Stranded Oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

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Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome

Genome Biology ◽

10.1186/s13059-021-02369-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Robin-Lee Troskie ◽

Yohaann Jafrani ◽

Tim R. Mercer ◽

Adam D. Ewing ◽

Geoffrey J. Faulkner ◽

...

Keyword(s):

Cultured Cells ◽

Open Reading Frames ◽

Cdna Sequencing ◽

Protein Coding ◽

Dynamic Component ◽

Gene Copies ◽

Long Read ◽

Normal Human ◽

Reading Frames ◽

Transcriptional Landscape

AbstractPseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.

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High Genetic Variability of the agr Locus in Staphylococcus Species

Journal of Bacteriology ◽

10.1128/jb.184.4.1180-1186.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 1180-1186 ◽

Cited By ~ 140

Author(s):

Philippe Dufour ◽

Sophie Jarraud ◽

Francois Vandenesch ◽

Timothy Greenland ◽

Richard P. Novick ◽

...

Keyword(s):

Signal Transduction ◽

Quorum Sensing ◽

Sequence Divergence ◽

Pcr Amplification ◽

Variable Region ◽

Cysteine Residue ◽

Multiple Alignment ◽

Open Reading Frames ◽

Signal Transduction System ◽

Reading Frames

ABSTRACT The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

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Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.1891-1899.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 1891-1899 ◽

Cited By ~ 42

Author(s):

Stephen McGrath ◽

Jos F. M. L. Seegers ◽

Gerald F. Fitzgerald ◽

Douwe van Sinderen

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Plasmid Vector ◽

Dna Interaction ◽

Open Reading Frames ◽

Phage Resistance ◽

High Copy Number Plasmid ◽

Specific Fragment ◽

Reading Frames

ABSTRACT A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

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The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria

Plant Disease ◽

10.1094/pdis-09-18-1561-re ◽

2019 ◽

Vol 103 (6) ◽

pp. 1302-1308 ◽

Cited By ~ 2

Author(s):

Nomatter Chingandu ◽

Lelia Dongo ◽

Osman A. Gutierrez ◽

Judith K. Brown

Keyword(s):

Phylogenetic Analyses ◽

Pcr Amplification ◽

West African ◽

Open Reading Frames ◽

Nucleotide Identity ◽

Genome Sequences ◽

Foliar Symptoms ◽

Field Samples ◽

Polymerase Chain ◽

Reading Frames

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.

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Genome-Scale Cloning and Expression of Individual Open Reading Frames Using Topoisomerase I-Mediated Ligation

Genome Research ◽

10.1101/gr.9.4.383 ◽

1999 ◽

Vol 9 (4) ◽

pp. 383-392

Author(s):

John A. Heyman ◽

Jeremiah Cornthwaite ◽

Luis Foncerrada ◽

Jeremiah R. Gilmore ◽

Erin Gontang ◽

...

Keyword(s):

Topoisomerase I ◽

Room Temperature ◽

Pcr Amplification ◽

Open Reading Frames ◽

Cloning And Expression ◽

Dna Topoisomerase ◽

Specific Restriction ◽

Genome Scale ◽

Reading Frames

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme’s high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

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FusoPortal: An interactive repository of hybrid MinION-sequenced Fusobacterium genomes improves gene identification and characterization

10.1101/305607 ◽

2018 ◽

Author(s):

Blake E. Sanders ◽

Ariana Umana ◽

Justin A. Lemkul ◽

Daniel J. Slade

Keyword(s):

Colorectal Cancer ◽

Molecular Mechanisms ◽

Open Reading Frames ◽

Reading Frame ◽

Genome Maps ◽

Complete Genomes ◽

Long Read ◽

Large Genes ◽

Identification And Characterization ◽

Reading Frames

AbstractHere we present FusoPortal, an interactive repository of Fusobacterium genomes that were sequenced using a hybrid MinION long-read sequencing pipeline, followed by assembly and annotation using a diverse portfolio of predominantly open-source software. Significant efforts were made to provide genomic and bioinformatic data as downloadable files, including raw sequencing reads, genome maps, gene annotations, protein functional analysis and classifications, and a custom BLAST server for FusoPortal genomes. FusoPortal has been initiated with eight complete genomes, of which seven were previously only drafts that varied from 24-67 contigs. We showcase that genomes in FusoPortal provide accurate open reading frame annotations, and have corrected a number of large genes (>3 kb) that were previously misannotated due to contig boundaries. In summary, FusoPortal (http://fusoportal.org) is the first database of MinION sequenced and completely assembled Fusobacterium genomes, and this central Fusobacterium genomic and bioinformatic resource will aid the scientific community in developing a deeper understanding of how this human pathogen contributes to an array of diseases including periodontitis and colorectal cancer.ImportanceIn this study, we report a hybrid MinION whole genome sequencing pipeline, and describe the genomic characteristics of the first eight strains deposited in the FusoPortal database. This collection of highly accurate and complete genomes drastically improves upon previous multi-contig assemblies by correcting and newly identifying a significant number of open reading frames. We believe this resource will result in the discovery of proteins and molecular mechanisms used by an oral pathogen, with the potential to further our understanding of how F. nucleatum contributes to a repertoire of diseases including periodontitis, pre-term birth, and colorectal cancer

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Forced Evolution Reveals the Importance of Short Open Reading Frame A and Secondary Structure in the Cauliflower Mosaic Virus 35S RNA Leader

Journal of Virology ◽

10.1128/jvi.72.5.4157-4169.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 4157-4169 ◽

Cited By ~ 29

Author(s):

Mikhail M. Pooggin ◽

Thomas Hohn ◽

Johannes Fütterer

Keyword(s):

Secondary Structure ◽

Mosaic Virus ◽

Pcr Amplification ◽

Cauliflower Mosaic Virus ◽

Open Reading Frames ◽

Reading Frame ◽

Short Open Reading Frame ◽

The Stability ◽

Virus Replication Cycle ◽

Reading Frames

ABSTRACT Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5′-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

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Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

10.1101/2019.12.13.876037 ◽

2019 ◽

Cited By ~ 2

Author(s):

Alexander M. Price ◽

Katharina E. Hayer ◽

Daniel P. Depledge ◽

Angus C. Wilson ◽

Matthew D. Weitzman

Keyword(s):

Rna Sequencing ◽

Open Reading Frames ◽

Short Read ◽

Sequencing Technologies ◽

Protein Functions ◽

Long Read ◽

Cleavage And Polyadenylation ◽

Polyadenylation Sites ◽

Splice Junctions ◽

Reading Frames

AbstractAdenovirus is a common human pathogen that relies on host cell processes for production and processing of viral RNA. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. The canonical viral early and late RNA cassettes were confirmed, but analysis of splice junctions within long RNA reads revealed an additional 20 novel viral transcripts. These RNAs include seven new splice junctions which lead to expression of canonical open reading frames (ORF), as well as 13 transcripts encoding for messages that potentially alter protein functions through truncations or the fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

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