hyRAD RNA probes preparation and capture v2

Supplemental Information for: Suchan, T., Kusliy, M.A., Khan, N., Chauvey, L., Tonasso-Calvière, L., Schiavinato, S., Southon, J., Keller, M., Kitagawa, K., Krause, J., Bessudnov, A.N., Bessudnov, A.A., Graphodatsky, A.S., Lamas, S.V., Wilczyński, J., Pospuła, S., Tunia, K., Nowak, M., Moskal-delHoyo, M., Tishkin, A.A., Pryor, A.J.E., Outram, A.K., Orlando, L. (2021) Performance and automation of ancient DNA capture with RNA hyRAD probes. Molecular Ecology Resources, doi: 10.1111/1755-0998.13518

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram- positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

Calibration-Less DNA Concentration Measurements Using EOF Volumetric Flow and Single Molecule Counting

Nanopore sensing is a promising tool well suited to capture and detect DNA and other single molecules. DNA is a negatively charged biomolecule that can be captured and translocated through a constricted nanopore aperture under an applied electric field. Precise assessment of DNA concentration is of crucial importance in many analytical processes and medical diagnostic applications. Recently, we found that hydrodynamic forces can lead to DNA motion against the electrophoretic force (EPF) at low ionic strength. This study utilized glass nanopores to investigate the DNA capture mechanism and detect DNA molecules due to volumetric flow at these low ionic strength conditions. We measured the DNA capture rate at five different pico-molar concentrations. Our findings indicated that the translocation rate is proportional to the concentration of DNA molecules and requires no calibration due to the volumetric flow rate and DNA counting directly correlates with concentration. Using finite element analysis, we calculated the volumetric flow and proposed a simple, straightforward approach for accurate DNA quantification. Furthermore, these experiments explore a unique transport mechanism where one of the most highly charged molecules enters a pore against electric field forces. This quantitative technique has the potential to provide distinct insight into nanopore-based biosensing and further enhance the nanopore’s capability as a biomolecule concentration sensor.

Specific High-sensitivity Multiple-probe-assisted DNA Capture and Amplification Technology for Direct Detection of African Swine Fever Virus without DNA Extraction

African Swine Fever (ASF) is one of the most devastating infectious diseases affecting domestic pigs and wild boar. The grave socio-economic impact of African Swine Fever infection at a global level makes large-scale rapid and robust diagnosis a critical step towards effective control. However, the nucleic acid purification required in most molecular detection methods is time- and labor-intensive, prone to nucleic acid loss or contamination, and impractical for massive active screening or for use in resource-limited areas. Here we describe multiple-probe-assisted DNA capture and amplification technology (MADCAT) - a novel sensitive, simple, and reliable method for detecting ASFV directly from whole blood or other complex matrices. Through the unique DNA capture method which specifically capture only the target DNA onto the well for subsequent amplification, MADCAT abandons the complicated extraction protocol and achieves ultrafast and high-throughput detection. The sample-to-result time for 96 samples is about 100 min, as compared with the 3 - 4 h time of the standard real time qPCR method. The limit of detection (LOD) is 0.5 copies/μL and is 10 times more sensitive than an OIE-recommended qPCR assay when testing serially diluted whole blood samples. The assay is 100% specific against other common swine pathogens. In clinical diagnosis of 48 field samples, all 22 positive samples were correctly identified with lower Ct values than OIE-recommended qPCR, confirming its high diagnostic sensitivity (100%). Owing to its high-throughput, specific high-sensitivity, and cost-efficient features, MADCAT shows great potential for future use in clinical ASFV active screening.

A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5′ end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

Discovery and Characterization of a Hidden Retroviral Enhancer by Viral DNA-capture-seq Approach

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes a cancer of infected cells called adult T-cell leukemia (ATL). There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active in vivo even in proviruses lacking the 5’ long terminal repeat (LTR), a known viral enhancer and promoter. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture followed by high-throughput sequencing, we found a previously unidentified viral enhancer not in the LTR but in the middle of the HTLV-1 provirus. The host transcription factors, SRF and ELK-1, bind to this enhancer region both in cell lines and in freshly isolated ATL cells. HTLV-1 containing mutations in the SRF- and ELK-1-binding sites markedly decreased chromatin openness at the viral enhancer, viral gene transcription, and enhancing effects on host gene transcription near the viral integration site. Aberrant host genome transcription was observed at nearby integration sites in defective proviruses containing the enhancer in ATL cells. This finding reveals how the exogenous retrovirus achieves persistent infection in the host via the internal viral enhancer and resolves certain long-standing questions concerning HTLV-1 infection. We anticipate that the DNA-capture-seq approach can be applied to analyze regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq) is a new ultra-sensitive UMI-based NGS library preparation technology for use with cfDNA and cfRNA

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20–25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9

CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.

Integrative and Conjugative Element ICETh1 Functions as a Pangenomic DNA Capture Module in Thermus thermophilus

Transjugation is an unconventional conjugation mechanism in Thermus thermophilus (Tth) that involves the active participation of both mating partners, encompassing a DNA secretion system (DSS) in the donor and an active natural competence apparatus (NCA) in the recipient cells. DSS is encoded within an integrative and conjugative element (ICETh1) in the strain Tth HB27, whereas the NCA is constitutively expressed in both mates. Previous experiments suggested the presence of multiple origins of transfer along the genome, which could generate genomic mosaicity among the progeny. Here, we designed transjugation experiments between two closely related strains of Tth with highly syntenic genomes, containing enough single nucleotide polymorphisms to allow precise parenthood analysis. Individual clones from the progeny were sequenced, revealing their origin as derivatives of our ICETh1-containing intended “donor” strain (HB27), which had acquired separate fragments from the genome of the ICETh1-free HB8 cells, which are our intended recipient. Due to the bidirectional nature of transjugation, only assays employing competence-defective HB27 derivatives as donors allowed the recovery of HB8-derived progeny. These results show a preference for a retrotransfer mechanism in transjugation in ICETh1-bearing strains, supporting an inter-strain gene-capture function for ICETh1. This function could benefit the donor-capable host by facilitating the acquisition of adaptive traits from external sources, ultimately increasing the open pangenome of Thermus, maximizing the potential repertoire of physiological and phenotypical traits related to adaptation and speciation.