monaLisa: an R/Bioconductor package for identifying regulatory motifs

Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R.AvailabilitymonaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/[email protected]

Biosynthesis and Transport of Nucleotide Sugars for Plant Hemicellulose

Hemicellulose is entangled with cellulose through hydrogen bonds and meanwhile acts as a bridge for the deposition of lignin monomer in the secondary wall. Therefore, hemicellulose plays a vital role in the utilization of cell wall biomass. Many advances in hemicellulose research have recently been made, and a large number of genes and their functions have been identified and verified. However, due to the diversity and complexity of hemicellulose, the biosynthesis and regulatory mechanisms are yet unknown. In this review, we summarized the types of plant hemicellulose, hemicellulose-specific nucleotide sugar substrates, key transporters, and biosynthesis pathways. This review will contribute to a better understanding of substrate-level regulation of hemicellulose synthesis.

Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data.

Variation in synonymous nucleotide composition among genomes of sarbecoviruses and consequences for the origin of COVID-19

The subgenus Sarbecovirus includes two human viruses, SARS-CoV and SARS-CoV-2, respectively responsible for the SARS epidemic and COVID-19 pandemic, as well as many bat viruses and two pangolin viruses.Here, the synonymous nucleotide composition (SNC) of Sarbecovirus genomes was analysed by examining third codon-positions, dinucleotides, and degenerate codons. The results show evidence for the eigth following groups: (i) SARS-CoV related coronaviruses (SCoVrC including many bat viruses from China), (ii) SARS-CoV-2 related coronaviruses (SCoV2rC; including five bat viruses from Cambodia, Thailand and Yunnan), (iii) pangolin viruses, (iv) three bat viruses showing evidence of recombination between SCoVrC and SCoV2rC genomes, (v) two highly divergent bat viruses from Yunnan, (vi) the bat virus from Japan, (vii) the bat virus from Bulgaria, and (viii) the bat virus from Kenya. All these groups can be diagnosed by specific nucleotide compositional features except the one concerned by recombination between SCoVrC and SCoV2rC. In particular, SCoV2rC genomes are characterised by the lowest percentages of cyosine and highest percentages of uracil at third codon-positions, whereas the genomes of pangolin viruses exhibit the highest percentages of adenine at third codon-positions. I suggest that latitudinal and taxonomic differences in the imbalanced nucleotide pools available in host cells during viral replication can explain the seven groups of SNC here detected among Sarbecovirus genomes. A related effect due to hibernating bats is also considered. I conclude that the two independent host switches from Rhinolophus bats to pangolins resulted in convergent mutational constraints and that SARS-CoV-2 emerged directly from a horseshoe bat virus.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing

Background Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. Results A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Intraspecific Genetic Distance and Phylogenetic Evolution of Schizothorax Plagiostomus Inferred from Mitochondrial D-Loop Sequences

The fish in the genus Schizothorax from the Cyprinidae family live in high-altitude Rivers andstreams, are threatened by various anthropogenic stressors. This study aims to characterize S. plagiostomus across Pakistan and throughout the world available on NCBI using the mitochondrial D-loop region, and in particular, to assess the degree of intra-specific pairwise distance among these sequences, as well as to establish their phylogenetic relationships. The percent overall nucleotide composition was 32.6% (A), 33.6% (T), 19.8% (C), and 14.0% (G), which infers that S. plagiostomus control regions is AT-rich (66.2%) and poor in G contents. The mean pair-wise intra-specific nucleotide diversity (Pi)of all the S. plagiostomus was 0.022. While, the inter-specific nucleotide diversity of all the Schizothorax species was 0.049. D-loop sequences for intra-specific variations revealed 765 sites were invariable and 10 were variable, 8 parsimony informative sites and only 2 were singletons. The overall transition/transversion ratio is R = 7.135. Three domains in S. plagiostomus were observed, namely, the termination associated sequence (TAS) domain, the central conserved sequence block (CSB) domain, and the conserved sequence block (CSB) domain. No substitution saturation was detected as an Iss value was significantly (𝑃< 0.001) lower than the Iss.c in all cases indicating the suitability of the data for phylogenetic analysis. This study signifies the importance of the control region for the genetic analysis of S. plagiostomus and also provides a hypothesis of their phylogenetic relationships.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus Group using quantitative sequencing

Background Vivax malaria is transmitted by members of the Anopheles Hyrcanus Group that includes six species (Anopheles sinensis s.s., An. pullus, An. kleini, An. belenrae, An. lesteri, and An. sineroides) in Republic of Korea. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species specific molecular markers requires extensive time commitment and financial resources. Results A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification markers in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different loci. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C markers at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T markers. A blind test proved that predicted species composition ratios were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay in place

Labelling strategies in metabarcoding studies &amp; how to ensure that nucleotide tags stay in place Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of ecological interactions, diet and biodiversity, as its labelling of amplicons allows high-throughput sequencing of taxonomically informative DNA sequences from many samples in parallel. The backbone of metabarcoding is the addition of sample-specific nucleotide identifiers to amplicons and then following sequencing using these to assign metabarcoding sequences to the samples they originated from. This allows the pooling of hundreds to thousands of samples before sequencing and thereby full utilisation of the capacity of high-throughput sequencing platforms. The nucleotide identifiers can be added both during the metabarcoding PCR and during library preparation, i.e. when amplicons are prepared for sequencing. There are three main strategies with which to achieve nucleotide labelling in metabarcoding studies. One commonly used strategy is the so-called tagged PCR approach in which DNA extracts are individually amplified with metabarcoding primers that carry sample-specific nucleotide tags at the 5’ end. The uniquely tagged products are then pooled and a library prepared on the pool of amplicons. However, tag‐jumps have been documented in this commonly used metabarcoding approach (Schnell et al. 2015). Tag-jumps cause nucleotide tags to switch between amplicons, resulting in occurrence of amplicons that carry different tags than originally applied. Sequences in the sequencing output that carry tag combinations not used in the study design are easily identified and excluded. However, sequences carrying incorrect, but already used, tag combinations will cause incorrect assignments of sequences to samples. This can - much to the detriment of metabarcoding studies - lead to false positives and artificial inflation of diversity in the samples (Schnell et al. 2015). The occurrence of tag-jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin-tags to ensure that tag‐jumps cannot result in false assignments of sequences to samples (Schnell et al. 2015). However, this increases both cost and workload of metabarcoding studies. In a recently published article, we demonstrate a tag-jump free single-tube library preparation protocol for Illumina sequencing specifically designed for 5’ nucleotide tagged amplicons, the Tagsteady protocol (Carøe &amp; Bohmann 2020). We designed the Tagsteady protocol to circumvent the two steps during library preparation of pools of 5ʹ nucleotide-tagged amplicons that had previously been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step, and ii) post-ligation PCR amplification of amplicon libraries. We used pools of twin‐tagged amplicons to investigate the effect of these two steps on the occurrence of tag‐jumps. Doing this, we demonstrated that blunt‐ending and post-ligation PCR, alone or together, can result in high proportions of tag-jumps, in our study up to ca. 49% of total sequences. The Tagsteady protocol where both these steps were left out resulted in tag‐jump levels comparable to background contamination (Carøe &amp; Bohmann 2020). In our study, we encourage practitioners to avoid using T4 DNA polymerase blunt‐ending and post-ligation PCR in library preparation of 5’ nucleotide tagged amplicon pools, for example by using the Tagsteady protocol (Carøe &amp; Bohmann 2020). This will enable efficient and cost-effective generation of metabarcoding data with correct assignment of sequences to samples. References Carøe C, Bohmann K (2020) Tagsteady: A metabarcoding library preparation protocol to avoid false assignment of sequences to samples. Molecular Ecology Resources, 20, 1620–1631. Schnell IB, Bohmann K, Gilbert MTP (2015) Tag jumps illuminated - reducing sequence-to-sample misidentifications in metabarcoding studies. Molecular Ecology Resources, 15, 1289–1303.

COMPREHENSIVE ANALYSIS OF THE GENOMES OF RUSSIAN CATTLE AND SHEEP BREEDS TO FIND NUCLEOTIDE POLYMORPHISMS TO IMPROVE ADAPTATION AND ECONOMICALLY IMPORTANT PHENOTYPES AT THE GENE LEVEL

Our work shows the need to apply an integrated approach to identify specific nucleotide polymorphisms that can be used to increase the efficiency and/or acclimatization of farm animal breeds in environmental conditions of Russia using genetic technologies.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

ABSTRACTBase editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors, and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.