Enabling inference for context-dependent models of mutation by bounding the propagation of dependency

Although the rates at which positions in the genome mutate are known to depend not only on the nucleotide to be mutated, but also on neighboring nucleotides, it remains challenging to do phylogenetic inference using models of context-dependent mutation. In these models, the effects of one mutation may in principle propagate to faraway locations, making it difficult to compute exact likelihoods. This paper shows how to use bounds on the propagation of dependency to compute likelihoods of mutation of a given segment of genome by marginalizing over sufficiently long flanking sequence. This can be used for maximum likelihood or Bayesian inference. Protocols examining residuals and iterative model refinement are also discussed. Tools for efficiently working with these models are provided in an R package, that could be used in other applications. The method is used to examine context dependence of mutations since the common ancestor of humans and chimpanzee.

Whole genome sequencing and characteristics of Escherichia coli with co-existence of ESBL and mcr genes from pigs

This study aimed to analyze three ESBL-producing E. coli co-harboring mcr and ESBL genes from a healthy fattening pig (E. 431) and two sick pigs (ECP.81 and ECP.82) in Thailand using Whole Genome Sequencing (WGS) using either Illumina MiSeq or HiSeq PE150 platforms to determine their genome and transmissible plasmids. E. 431 carrying mcr-2.1 and mcr-3.1 belonged to serotype O142:H31 with ST29 sequence type. ECP.81 and ECP.82 from sick pigs harboring mcr-1.1 and mcr-3.1 were serotype O9:H9 with ST10. Two mcr-1.1 gene cassettes from ECP.81 and ECP.82 were located on IncI2 plasmid with 98% identity to plasmid pHNSHP45. The mcr-2.1-carrying contig in E. 431 showed 100% identity to plasmid pKP37-BE with the upstream flanking sequence of IS1595. All three mcr-3.1-carrying contigs contained the ΔTnAs2-mcr-3.1-dgkA core segment and had high nucleotide similarity (85–100%) to mcr-3.1-carrying plasmid, pWJ1. The mobile elements i.e. IS4321, ΔTnAs2, ISKpn40 and IS3 were identified in the flanking regions of mcr-3. Several genes conferring resistance to aminoglycosides (aac(3)-IIa, aadA1, aadA2b, aph(3’’)-Ib, aph(3’)-IIa and aph(6)-Id), macrolides (mdf(A)), phenicols (cmlA1), sulphonamide (sul3) and tetracycline (tet(A) and tet(M)) were located on plasmids, of which their presence was well corresponded to the host’s resistance phenotype. Amino acid substitutions S83L and D87G in GyrA and S80I and E62K in ParC were observed. The blaCTX-M-14 and blaCTX-M-55 genes were identified among these isolates additionally harbored blaTEM-1B. Co-transfer of mcr-1.1/blaTEM-1B and mcr-3.1/blaCTX-M-55 was observed in ECP.81 and ECP.82 but not located on the same plasmid. The results highlighted that application of advanced innovation technology of WGS in AMR monitoring and surveillance provide comprehensive information of AMR genotype that could yield invaluable benefits to development of control and prevention strategic actions plan for AMR.

Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold

Guanine-quadruplex-based CpG oligodeoxynucleotides (G4 CpG ODNs) have been developed as potent immunostimulatory agents with reduced sensitivity to nucleases. We designed new monomeric G4 ODNs with an antiparallel topology using antiparallel type duplex/G4 ODNs as robust scaffolds, and we characterized their topology and effects on cytokine secretion. Based on circular dichroism analysis and quantification of mRNA levels of immunostimulatory cytokines, it was found that monomeric antiparallel G4 CpG ODNs containing two CpG motifs in the first functional loop, named G2.0.0, could maintain antiparallel topology and generate a high level of immunostimulatory cytokines in RAW264 mouse macrophage-like cell lines. We also found that the flanking sequence in the CpG motif altered the immunostimulatory effects. Gc2c.0.0 and Ga2c.0.0 are monomeric antiparallel G4 CpG ODNs with one cytosine in the 3′ terminal and one cytosine/adenine in the 5′ terminal of CpG motifs that maintained the same resistance to degradation in serum as G2.0.0 and improved interleukin-6 production in RAW264 and bone marrow-derived macrophages. The immunostimulatory activity of antiparallel G4 CpG ODNs is superior to that of linear natural CpG ODNs. These results provide insights for the rational design of highly potent CpG ODNs using antiparallel G4 as a robust scaffold.

DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of enhancers

Enhancer sequences control gene expression and comprise binding sites (motifs) for different transcription factors (TFs). Despite extensive genetic and computational studies, the relationship between DNA sequence and regulatory activity is poorly understood and enhancer de novo design is considered impossible. Here we built a deep learning model, DeepSTARR, to quantitatively predict the activities of thousands of developmental and housekeeping enhancers directly from DNA sequence in Drosophila melanogaster S2 cells. The model learned relevant TF motifs and higher-order syntax rules, including functionally non-equivalent instances of the same TF motif that are determined by motif-flanking sequence and inter-motif distances. We validated these rules experimentally and demonstrated their conservation in human by testing more than 40,000 wildtype and mutant Drosophila and human enhancers. Finally, we designed and functionally validated synthetic enhancers with desired activities de novo.

Flanker: a tool for comparative genomics of gene flanking regions

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial-resistance genes. Here, we present Flanker, a Python package that performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as ‘flank patterns’ (FPs), are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterized to fine-tune outputs by characterizing upstream and downstream regions separately, and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (bla OXA-48 and bla KPC-2/3) and show that it successfully identifies distinct clusters of FPs, including both known and previously uncharacterized structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterized using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking-gene characterization. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and bla OXA-48 upregulation and demonstrate 17 FPs (compared to the 5 previously identified). More generally, the demonstration in this study that FPs are associated with geographical regions and antibiotic-susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

indelPost: harmonizing ambiguities in simple and complex indel alignments

Summary Small insertions and deletions (indels) in nucleotide sequence may be represented differently between mapping algorithms and variant callers, or in the flanking sequence context. Representational ambiguity is especially profound for complex indels, complicating comparisons between multiple mappings and call sets. Complex indels may additionally suffer from incomplete allele representation, potentially leading to critical misannotation of variant effect. We present indelPost, a Python library that harmonizes these ambiguities for simple and complex indels via realignment and read-based phasing. We demonstrate that indelPost enables accurate analysis of ambiguous data and can derive the correct complex indel alleles from the simple indel predictions provided by standard small variant detectors, with improved performance over a specialized tool for complex indel analysis. Availability indelPost is freely available at: https://github.com/stjude/indelPost. Supplementary information Supplementary data are available at Bioinformatics online.

Nearest-neighbor effects modulate loxP spacer DNA chemical shifts and guide oligonucleotide design for NMR studies

Cre recombinase catalyzes site-specific DNA recombination at pseudo-palindromic loxP sites through two rounds of strand cleavage, exchange, and religation. Cre is a potential gene editing tool of interest due its lack of requirements for external energy sources or host factors, as well as the fact that it does not generate potentially cytotoxic double-stranded DNA breaks. However, broader applications of Cre in editing noncanonical target sequences requires a deeper understanding of the DNA features that enable target site selection and efficient recombination. Although Cre recombines loxP DNA in a specific and ordered fashion, it makes few direct contacts to the loxP spacer, the region where recombination occurs. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. We report sequential backbone resonance assignments for loxP oligonucleotides of varying lengths and evaluate chemical shift differences, Δδ, between the oligos. Analysis of flanking sequence effects and mutations on spacer chemical shifts indicates that nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that adequately preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

Background Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.