Genomic Fishing and Data Processing for Molecular Evolution Research.

Molecular evolution analyses, such as detection of adaptive/purifying selection or ancestral protein reconstruction, typically require three inputs for a target gene (or gene family) in a particular group of organisms: sequence alignment, model of evolution, and phylogenetic tree. While modern advances in high-throughput sequencing techniques have led to rapid accumulation of genomic-scale data in public repositories and databases, mining such vast amount of information often remains a challenging enterprise. Here, we describe a comprehensive, versatile workflow aimed at the preparation of genome-extracted datasets readily available for molecular evolution research. The workflow involves: (1) fishing (searching and capturing) specific gene sequences of interest from taxonomically diverse genomic data available in databases at variable levels of annotation, (2) processing and depuration of retrieved sequences, (3) production of a multiple sequence alignment, (4) selection of best-fit model of evolution, and (5) solid reconstruction of a phylogenetic tree.

Variable number tandem repeats of a 9-base insertion in the N-terminal domain of severe acute respiratory syndrome coronavirus 2 spike gene.

Introduction: The world is still struggling against the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in 2022. The pandemic has been facilitated by the intermittent emergence of variant strains, which has been explained and classified mainly by the patterns of point mutations of the spike (S) gene. However, the profiles of insertions/deletions (indels) in SARS-CoV-2 genomes during the pandemic remain largely unevaluated yet. Methods: In this study, we first screened for the genome regions of polymorphic indel sites by performing multiple sequence alignment; then, NCBI BLAST search and GISAID database search were performed to comprehensively investigate the indel profiles at the polymorphic indel hotspot and elucidate the emergence and spread of the indels in time and geographical distribution. Results: A polymorphic indel hotspot was identified in the N-terminal domain of the S gene at approximately 22,200 nucleotide position, corresponding to 210-215 amino acid positions of SARS-CoV-2 S protein. This polymorphic hotspot was comprised of adjacent 3-base deletion (5'-ATT-3'; Spike_N211del) and 9-base insertion (5'-AGCCAGAAG-3'; Spike_ins214EPE). By performing NCBI BLAST search and GISAID database search, we identified several types of tandem repeats of the 9-base insertion, creating an 18-base insertion (Spike_ins214EPEEPE, Spike_ins214EPDEPE). The results of the searches suggested that the two-cycle tandem repeats of the 9-base insertion were created in November 2021 in Central Europe, whereas the emergence of the original one-cycle 9-base insertion (Spike_ins214EPE) would date back to the middle of 2020 and was away from the Central Europe. The identified 18-base insertions based on 2-cycle tandem repeat of the 9-base insertion were collected between November 2021 and April 2022, suggesting that these mutations could not survive and have been already eliminated. Discussion: The GISAID database search implied that this polymorphic indel hotspot to be with one of the highest tolerability for incorporating indels in SARS-CoV-2 S gene. In summary, the present study identified a variable number of tandem repeat of 9-base insertion in the N-terminal domain of SARS-CoV-2 S gene, and the repeat could have occurred at different time from the insertion of the original 9-base insertion.

Searching for remote homologs of CAML among eukaryotes.

The tryptophan rich basic protein/calcium signal-modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail-anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous. CAML contains an N-terminal basic, TRC40/Get3-interacting, region, three transmembrane segments near the C-terminus, and a poorly conserved region between these domains. Here, I searched the NCBI protein database for remote CAML homologs in all eukaryotes, using position-specific iterated-basic local alignment search tool, with the C-terminal, the N-terminal or the full-length sequence of human CAML as query. The N-terminal basic region and full-length CAML retrieved homologs among metazoa, plants and fungi. In the latter group several hits were annotated as GET2. The C-terminal query did not return entries outside of the animal kingdom, but did retrieve over one hundred invertebrate metazoan CAML-like proteins, which all conserved the N-terminal TRC40-binding domain. The results indicate that CAML homologs exist throughout the eukaryotic domain of life, and suggest that metazoan CAML and yeast GET2 share a common evolutionary origin. They further reveal a tight link between the particular features of the metazoan membrane-anchoring domain and the TRC40-interacting region. The list of sequences presented here should provide a useful resource for future studies addressing structure-function relationships in CAML proteins.

Zika Virus Amplification Using Strand Displacement Isothermal Method and Sequencing Using Nanopore Technology.

Development of novel point of care diagnostic methods in order to help in implementing disease control program and identifying the causative agent of an outbreak is crucial. Classical diagnostic techniques, e.g., real-time polymerase chain reaction (PCR), rely on the presence of the nucleic acid sequence of the target in GenBank. In the case of an emerging new strain of a known or novel pathogen, false-negative results will be recorded by PCR. On the other hand, next-generation sequencing technologies allow rapid whole genome sequencing without previous knowledge of the target. One of these methods is the Oxford Nanopore sequencing technique, which utilizes a portable device named MinION and has a short run time. In this protocol, we describe the development of a novel nanopore sequencing protocol by combining random isothermal amplification technology and nanopore sequencing. The established protocol is rapid (<7 h) and sensitive as less than 4% of the sequenced RNA belonged to the target virus, Zika. Interestingly, we have established an offline BLAST search for the data analysis that facilitates the use of the whole protocol at remote settings without the need of an Internet connection.

Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization.

Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5'-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5'-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories.

CABRA: Cluster and Annotate Blast Results Algorithm.

BACKGROUND: Basic local alignment search tool (BLAST) searches are frequently used to look for homologous sequences and to annotate a query protein, but the increasing size of protein databases makes it difficult to review all results from a similarity search. FINDINGS: We developed a web tool called Cluster and Annotate Blast Results Algorithm (CABRA), which enables a rapid BLAST search in a variety of updated reference proteomes, and provides a new way to functionally evaluate the results by the subsequent clustering of the hits and annotation of the clusters. The tool can be accessed from the following web-resource: http://cbdm-01.zdv.uni-mainz.de/~munoz/CABRA . CONCLUSIONS: Cluster and Annotate Blast Results Algorithm simplifies the analysis of the results of a BLAST search by providing an overview of the result's annotations organized in clusters that can be iteratively modified by the user.

Chromosomal localization of microsatellite loci in Drosophila mediopunctata.

Drosophila mediopunctata has been used as a model organism for genetics and evolutionary studies in the last three decades. A linkage map with 48 microsatellite loci recently published for this species showed five syntenic groups, which had their homology determined to Drosophila melanogaster chromosomes. Then, by inference, each of the groups was associated with one of the five major chromosomes of D. mediopunctata. Our objective was to carry out a genetic (chromosomal) analysis to increase the number of available loci with known chromosomal location. We made a simultaneous analysis of visible mutant phenotypes and microsatellite genotypes in a backcross of a standard strain and a mutant strain, which had each major autosome marked. Hence, we could establish the chromosomal location of seventeen loci; including one from each of the five major linkage groups previously published, and twelve new loci. Our results were congruent with the previous location and they open new possibilities to future work integrating microsatellites, chromosomal inversions, and genetic determinants of physiological and morphological variation.

A genome-wide identification of basic helix-loop-helix motifs in Pediculus humanus corporis (Phthiraptera: Pediculidae).

Basic helix-loop-helix (bHLH) proteins comprise a large superfamily of transcription factors, which are involved in the regulation of various developmental processes. bHLH family members are widely distributed in various eukaryotes including yeast, fruit fly, zebrafish, mouse, and human. In this study, we identified 55 bHLH motifs encoded in genome sequence of the human body louse, Pediculus humanus corporis (Phthiraptera: Pediculidae). Phylogenetic analyses of the identified P. humanus corporis bHLH (PhcbHLH) motifs revealed that there are 23, 11, 9, 1, 10, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Examination to GenBank annotations of the 55 PhcbHLH members indicated that 29 PhcbHLH proteins were annotated in consistence with our analytical result, 8 were annotated different with our analytical result, 12 were merely annotated as hypothetical protein, and the rest 6 were not deposited in GenBank. A comparison on insect bHLH gene composition revealed that human body louse possibly has more hairy and E(spl) genes than other insect species. Because hairy and E(spl) genes have been found to negatively regulate the differentiation of insect preneural cells, it is suggested that the existence of additional hairy and E(spl) genes in human body louse is probably the consequence of its long period adaptation to the relatively dark and stable environment. These data provide good references for further studies on regulatory functions of bHLH proteins in the growth and development of human body louse.

A genome-wide identification and classification of basic helix-loop-helix genes in the jewel wasp, Nasonia vitripennis (Hymenoptera: Pteromalidae).

Basic helix-loop-helix (bHLH) proteins are highly conserved DNA-binding transcription factors of a large superfamily. Animal bHLH proteins play important regulatory roles in various developmental processes such as neurogenesis, myogenesis, heart development, and hematopoiesis. The jewel wasp (Nasonia vitripennis) is a good model organism of hymenoptera insects for studies of developmental and evolutionary genetics. In this study, we identified 48 bHLH genes in the genome of N. vitripennis. According to phylogenetic analysis, based on N. vitripennis bHLH (NvbHLH) motif sequences and structural domain distribution in their full-length protein sequences, the identified NvbHLH genes were classified into 36 bHLH families with 19, 12, 9, 1, 6, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Our classification to the identified NvbHLH family members confirms GenBank annotations for 21 of the 48 NvbHLH proteins and provides useful information for further characterization and annotation of the remaining 27 NvbHLH proteins. Compared to other insect species, N. vitripennis has the lowest number of bHLH family members. No NvbHLH members have been found in the families Net, MyoRa, and PTFa, while all other insect species have at least one member in each of the families. These data constitute a solid basis for further investigations into the functions of bHLH proteins in developmental regulation of N. vitripennis.

HPV-QUEST: A highly customized system for automated HPV sequence analysis capable of processing Next Generation sequencing data set.

Next Generation sequencing (NGS) applied to human papilloma viruses (HPV) can provide sensitive methods to investigate the molecular epidemiology of multiple type HPV infection. Currently a genotyping system with a comprehensive collection of updated HPV reference sequences and a capacity to handle NGS data sets is lacking. HPV-QUEST was developed as an automated and rapid HPV genotyping system. The web-based HPV-QUEST subtyping algorithm was developed using HTML, PHP, Perl scripting language, and MYSQL as the database backend. HPV-QUEST includes a database of annotated HPV reference sequences with updated nomenclature covering 5 genuses, 14 species and 150 mucosal and cutaneous types to genotype blasted query sequences. HPV-QUEST processes up to 10 megabases of sequences within 1 to 2 minutes. Results are reported in html, text and excel formats and display e-value, blast score, and local and coverage identities; provide genus, species, type, infection site and risk for the best matched reference HPV sequence; and produce results ready for additional analyses.

Expressed sequence tags analysis of Blattella germanica

Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About 42% (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling pathways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these represent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.