Analysis Of Amino Acid Variations Research Articles

Complete Genome Sequence and Construction of an Infectious Bacterial Artificial Chromosome Clone of a Virulent Duck Enteritis Virus Strain XJ

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.

Epidemiological and genetic characteristics of foot-and-mouth disease virus in China from 2010 to 2022

Foot-and-mouth disease virus (FMDV) is a highly contagious picornavirus that can infect cloven-hoofed animals of significant agricultural importance. In China, foot-and-mouth disease (FMD) epidemics occur annually, resulting in localized outbreaks or sporadic epidemics that cause significant economic losses. This study summarized 123 cases of FMD reported in China between 2010 and 2022, using data from the official website of the Chinese Center for Animal Disease Control and Prevention. The epidemic situation and genetic characteristics of FMDV in China were studied through phylogenetic analysis, amino acid variation analysis of antigenic epitopes, and genetic recombination analysis. The findings provide important references for predicting the FMDV epidemic situation in China, developing vaccines, and effectively preventing and controlling FMD.

Genetic Characteristics and Phylogeographic Dynamics of Echovirus.

Echoviruses belong to the genus Enterovirus in the Picornaviridae family, forming a large group of Enterovirus B (EV-B) within the Enteroviruses. Previously, Echoviruses were classified based on the coding sequence of VP1. In this study, we performed a reliable phylogenetic classification of 277 sequences isolated from 1992 to 2019 based on the full-length genomes of Echovirus. In this report, phylogenetic, phylogeographic, recombination, and amino acid variability landscape analyses were performed to reveal the evolutional characteristics of Echovirus worldwide. Echoviruses were clustered into nine major clades, e.g., G1-G9. Phylogeographic analysis showed that branches G2-G9 were linked to common strains, while the branch G1 was only linked to G5. In contrast, strains E12, E14, and E16 clustered separately from their G3 and G7 clades respectively, and became a separate branch. In addition, we identified a total of 93 recombination events, where most of the events occurred within the VP1-VP4 coding regions. Analysis of amino acid variation showed high variability in the a positions of VP2, VP1, and VP3. This study updates the phylogenetic and phylogeographic information of Echovirus and indicates that extensive recombination and significant amino acid variation in the capsid proteins drove the emergence of new strains.

A long-term retrospective analysis of the haemorrhagic fever with renal syndrome epidemic from 2005 to 2021 in Jiangxi Province, China

Jiangxi is one of the provinces in China most seriously affected by the haemorrhagic fever with renal syndrome (HFRS) epidemic. The aim of this paper was to systematically explore the HFRS epidemic in Jiangxi from the perspective of Hantavirus (HV) prevalence in rodents and humans and virus molecular characteristics. Individual information on all HFRS cases in Jiangxi from 2005 to 2021 was extracted from the China Information System for Disease Control and Prevention. All S and M fragment sequences of the Seoul virus and Hantan virus strains uploaded by Jiangxi and its neighbouring provinces and some representative sequences from provinces in China or some countries of Southeast Asia with the highest HV prevalence were retrieved and downloaded from NCBI GenBank. Periodogram and spatial autocorrelation were adopted for temporal periodicity and spatial clustering analysis of the HFRS epidemic. Joinpoint regression was utilized to explore the changing morbidity trend patterns of HFRS. Multiple sequence alignment and amino acid variation analysis were used to explore the homology and variation of strain prevalence in Jiangxi. Based on monthly morbidity time series, the periodogram analysis showed that the prevalence of HFRS had periodicities of 6 months and 12 months. Spatial autocorrelation analysis showed that HFRS distributed in Jiangxi was not random, with a “High-High” clustering area around Gaoan County. HFRS morbidity among the 0 ~ 15-year-old and ~ 61-year-old or older populations in Jiangxi increased significantly during the period of 2008–2015. Generally, HFRS morbidity was significantly positively correlated with the index of rat with virus (IRV) (r = 0.742) in the counties surrounding Gaoan from 2005 to 2019. HTNV strains in Jiangxi were in one independent branch, while the SEOV strains in Jiangxi were relatively more diverse. Both the YW89-15 and GAW30/2021 strains shared approximately 85% nucleotide homology and approximately 97% amino acid homology with their corresponding standard strains and vaccine strains. GAW30/2021 and YW89-15 had some amino acid site variations in nucleoprotein, glycoprotein precursor and RNA-dependent polymerase with their corresponding vaccine strains Z10 (HTNV) and Z37 (SEOV). The HFRS epidemic in Jiangxi has obvious temporal periodicity and spatial clustering, and the significant increase in the non-Immunization Expanded Program (EPI) targeted population (children and elderly) suggests that HFRS vaccination in this population needs to be considered. Although applying the EPI played a certain role in curbing the incidence of HFRS in Jiangxi from the perspective of ecological epidemiology, HTNV and SEOV strains prevalent in Jiangxi have some amino acid site variations compared to their corresponding vaccine strains, suggesting that HV variation needs to be continuously monitored in the future to observe vaccine protective efficiency.

A genetic barcode of SARS-CoV-2 for monitoring global distribution of different clades during the COVID-19 pandemic

ObjectiveThe SARS-CoV-2 pathogen has established endemicity in humans. This necessitates the development of rapid genetic surveillance methodologies to serve as an adjunct with existing comprehensive, albeit though slower, genome sequencing-driven approaches. MethodsA total of 21,789 complete genomes were downloaded from GISAID on May 28, 2020 for analyses. We have defined the major clades and subclades of circulating SARS-CoV-2 genomes. A rapid sequencing-based genotyping protocol was developed and tested on SARS-CoV-2-positive RNA samples by next-generation sequencing. ResultsWe describe 11 major mutations which defined five major clades (G614, S84, V251, I378 and D392) of globally circulating viral populations. The clades can specifically identify using an 11-nucleotide genetic barcode. An analysis of amino acid variation in SARS-CoV-2 proteins provided evidence of substitution events in the viral proteins involved in both host entry and genome replication. ConclusionGlobally circulating SARS-CoV-2 genomes could be classified into 5 major clades based on mutational profiles defined by an 11-nucleotide barcode. We have successfully developed a multiplexed sequencing-based, rapid genotyping protocol for high-throughput classification of major clade types of SARS-CoV-2 in clinical samples. This barcoding strategy will be required to monitor decreases in genetic diversity as treatment and vaccine approaches become widely available.

Amino acid variation analysis of surface spike glycoprotein at 614 in SARS-CoV-2 strains

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to disperse globally with worrisome speed, identifying amino acid variations in the virus could help to understand the characteristics of it. Here, we studied 489 SARS-CoV-2 genomes obtained from 32 countries from the Nextstrain database and performed phylogenetic tree analysis by clade, country, and genotype of the surface spike glycoprotein (S protein) at site 614. We found that virus strains from mainland China were mostly distributed in Clade B and Clade undefined in the phylogenetic tree, with very few found in Clade A. In contrast, Clades A2 (one case) and A2a (112 cases) predominantly contained strains from European regions. Moreover, Clades A2 and A2a differed significantly from those of mainland China in age of infected population (P = 0.0071, mean age 40.24 to 46.66), although such differences did not exist between the US and mainland China. Further analysis demonstrated that the variation of the S protein at site 614 (QHD43416.1: p.614D>G) was a characteristic of stains in Clades A2 and A2a. Importantly, this variation was predicted to have neutral or benign effects on the function of the S protein. In addition, global quality estimates and 3D protein structures tended to be different between the two S proteins. In summary, we identified different genomic epidemiology among SARS-CoV-2 strains in different clades, especially in an amino acid variation of the S protein at 614, revealing potential viral genome divergence in SARS-CoV-2 strains.

Analysis of amino acid variations of major proteins from severe acute respiratory syndrome coronavirus 2

Objective To identify key amino acid variations of major proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by biophysical methods. Methods Through amino acid sequence alignment, classification of variant amino acid residues, three-dimensional structure reconstruction of proteins, and electrostatic interaction analysis of amino acid residues, the key amino acid variations of major proteins from SARS-CoV-2 was analyzed with RaTG13, the bat coronavirus with the highest homology, as the reference. Results At least ten amino acid variations that affect the possible electrostatic interactions were identified in RNA-dependent RNA polymerase (RdRp), exoribonuclease (ExoN), uridylate-specific endoribonuclease (NendoU), and spike (S) protein from SARS-CoV-2. These variations may affect the spatial conformation and biological functions of the proteins. Conclusion The key amino acid variations of the major proteins from SARS-CoV-2 have been preliminarily identified, providing clues for understanding the genetic, pathogenic and epidemiological characteristics of the virus..

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics.

BACKGROUND The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes.OBJECTIVES To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development.METHODS We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses.FINDINGS The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence.MAIN CONCLUSIONS We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Mutational analysis of HIV-1 viral protein U at Ser52 and Ser56 among the HIV-1 infected patients of Manipur

BackgroundPhosphorylation by Casein kinase 2 at two conserved seryl residues, Ser52 and Ser56 at the cytoplasmic domain of HIV-1 Viral protein U (Vpu) are essential for inducing CD4 degradation. This study is conducted to investigate the variability of HIV-1 viral protein unique particularly at amino-acid position Ser52 and Ser56 among the HIV infected individuals in the northeastern region of India. Materials and methodsBlood samples from 90 HIV-1 infected patients of Manipur have been studied by viral amplification and sequencing at vpu gene of HIV-1. For analysis of amino acid variation at Ser52 and Ser56, nucleic acid sequences were translated into amino acid and aligned with reference strain of HIV-1. Phylogenetic tree was also inferred among the studied samples. ResultsThe results revealed that 93% of HIV-1 infected individuals harbored virus with conserved serine at both amino acid positions 52 and 56 of vpu which were known to have the ability to induce CD4 degradation while 1% harbored viral mutation at both Ser52 and Ser56, replaced by asparagine which has been predicted to may have lost the ability to induce CD4 degradation. Moreover, 5% and 1% of HIV-1 infected individuals were found to be infected with mutated virus at ser52 and ser56 of vpu respectively. ConclusionThe overall finding of this study reveals that seryl residue of vpu at Ser52 and Ser56 was highly conserved among the HIV-1 infected individuals of the study population.

Influence of amino acid variations in the NS3, NS4A and NS4B of HCV genotypes 1a, 1b, 3a, 3b and 6f on the response to pegylated interferon and ribavirin combination therapy

BackgroundIt has been suggested that HCV proteins, core, NS3/4A, NS4B, and NS5A, contribute to the resistance of HCV to IFN and ribavirin (RBV) treatments. AimTo assess the effects of HCV amino acid variations in NS3, NS4A and NS4B of HCV subtypes 1a, 1b, 3a, 3b and 6f on the response to pegylated interferon (Peg-IFN) and RBV therapy. MethodsOne hundred and thirty four HCV isolates of genotypes 1a, 1b, 3a, 3b and 6f obtained from HCV patients both before and at week 4 of treatments were evaluated. Amino acid sequences of NS3, NS4A and NS4B were analyzed and in compared to reference sequences of corresponding genotypes. ResultsThe data revealed that amino acid variations within the full-length NS3, protease and helicase domains of NS3 of HCV 1a from responders were significantly higher than those from treatment failure groups as compared to reference sequences of each corresponding genotype. Similar results were observed in the full-length and helicase domain but not in the protease domain of HCV 1b. However, the number of amino acid variations in NS3 of HCV 3a, 3b and 6f as well as in NS4A and NS4B showed no difference between the viruses from responders and treatment failure group. Analysis of amino acid variations both before and at week 4 of treatment revealed that the mean number of amino acid variation in the full-length NS3 of HCV 3a and 3b from responders were also significantly higher than those from the treatment failure group. ConclusionOur study suggests that the increase of amino acid variations within the NS3 protein of HCV 1a, 1b, 3a and 3b were associated with the response to Peg-IFN and RBV treatment in Thai patients.

The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003–2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003–2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.

Proteome-wide Analysis of Amino Acid Variations That Influence Protein Lysine Acetylation

Next-generation sequencing (NGS) technologies are yielding ever higher volumes of genetic variation data. Given this large amount of data, it has become both a possibility and a priority to determine what the functional implication of genetic variations is. Considering the essential roles of acetylation in protein functions, it is highly likely that acetylation related genetic variations change protein functions. In this work, we performed a proteome-wide analysis of amino acid variations that could potentially influence protein lysine acetylation characteristics in human variant proteins. Here, we defined the AcetylAAVs as acetylation related amino acid variations that affect acetylation sites or their interacting acetyltransferases, and categorized three types of AcetylAAVs. Using the developed prediction system, named KAcePred, we detected that 50.87% of amino acid variations are potential AcetylAAVs and 12.32% of disease mutations could result in AcetylAAVs. More interestingly, from the statistical analysis, we found that the amino acid variations that directly create new potential lysine acetylation sites have more chance to cause diseases. It can be anticipated that the analysis of AcetylAAVs might be useful to screen important polymorphisms and help to identify the mechanism of genetic diseases. A user-friendly web interface for analysis of AcetylAAVs is now freely available at http://bioinfo.ncu.edu.cn/AcetylAAVs_Home.aspx .

Genetic variation and phylogenetic analysis of porcine circovirus type 2 infections in central China

Porcine circovirus type 2 (PCV2) infection causes postweaning multisystemic wasting syndrome and porcine circovirus-associated diseases in many regions. A total of 77 sequences, including 31 sampled from Henan province of China, were retrieved from GenBank and subjected to amino acid variation and phylogenetic analyses. The two PCV genotypes prevailing in Henan were PCV-2a and PCV-2b with PCV-2b accounting for 93.5% (29/31) of the Henan isolates. The 31 Henan isolates all shared between 92.7 and 100% sequence similarity. Amino acid variation analysis of the capsid protein revealed that Henan PCV2 strains tended to accumulate more substitutions within epitopic regions-a substitution pattern consistent with host immune system-mediated selection of virus immune escape variants. The analysed PCV sequences carry evidence of at least six unique recombination events. Selective pressure analysis of the relative recombination-free ORF2 sequences of these viruses revealed evidence of sites that are likely evolving in response to host-driven immune pressures-a finding that coupled with information on the prevalent diversity in Henan PCV2 isolates of known immunoreactive genomic loci will aid in future studies aiming to assess the evolutionary responses of PCV2 in China to the widespread deployment of anti-PCV vaccines in the country.

Sequence analysis of gag-pol gene of HIV-1 B/C recombinant viruses in China

To characterize full length gag gene and partial pol gene of Chinese prevalent HIV-1 B/C recombinant strains, to explore the genetic difference between parent strains and B/C recombinant strains, and to investigate the mechanism of different biologic phenotype among them. The peripheral blood samples were collected from 138 HIV-positive persons from 12 regions of China respectively. Samples of total DNA were extracted from the peripheral blood mononuclear cells to undergo nested PCR and sequencing. The 117 HIV-1 CRF07-BC strains and 21 HIV-1 CRF08-BC strains were screened with the resulted that 5 CRF07-BC samples from Xinjiang and 1 CRF08-BC sample from Chongqing were regarded as the most potential new type recombinant viruses. The sequences thus obtained underwent phylogenetic tree analysis and amino acid variation analysis. Simplot software was used to analyze the sequence recombination and identify the breakpoints of B/C recombinant strains. To confirm the breakpoints, separate phylogenetic analysis according to the breakpoints was performed with MEGA software. The genetic distances of different gene fragments were calculated by DISTANCE program in GCG software package. The gene dispersion in the gene fragment of a length of 2550 bp of the recombinant HIV-1-B/C and the potential influence of gene recombination on its function were further analyzed. No change of breakpoint was found in the 5 samples from Xinjiang. But a breakpoint shift of 160 nucleotides occurred in RT region of a sample from Congqing city. CRF07-BC and CRF08-BC remain the main prevalent HIV-1 B/C recombinant strains in China. No epidemic of new mosaic recombinant strain is found. The variation of amino acids at the sites 286 and 799 may be the reasons of the transmission dominance of the B/C recombinant strain.

Mapping structural determinants of biological activities in snake venom phospholipases A 2 by sequence analysis and site directed mutagenesis

In addition to their catalytic activity, snake venom phospholipases A 2 (vPLA 2) present remarkable diversity in their biological effects. Sequence alignment analyses of functionally related PLA 2 are frequently used to predict the structural determinants of these effects, and the predictions are subsequently evaluated by site directed mutagenesis experiments and functional assays. In order to improve the predictive potential of computer-based analysis, a simple method for scanning amino acid variation analysis (SAVANA) has been developed and included in the analysis of the lysine 49 PLA 2 myotoxins (Lys49-PLA 2). The SAVANA analysis identified positions in the C-terminal loop region of the protein, which were not identified using previously available sequence analysis tools. Site directed mutagenesis experiments of bothropstoxin-I, a Lys49-PLA 2 isolated from the venom of Bothrops jararacussu, reveals that these residues are exactly those involved in the determination of myotoxic and membrane damaging activities. The SAVANA method has been used to analyse presynaptic neurotoxic and anti-coagulant vPLA 2s, and the predicted structural determinants of these activities are in excellent agreement with the available results of site directed mutagenesis experiments. The positions of residues involved in the myotoxic and neurotoxic determinants demonstrate significant overlap, suggesting that the multiple biological effects observed in many snake vPLA 2s are a consequence of superposed structural determinants on the protein surface.

137 VX-950: a tight-binding HCV protease inhibitor with a superior sustained inhibitory response in HCV replicon cells

It has been suggested that HCV proteins, core, NS3/4A, NS4B, and NS5A, contribute to the resistance of HCV to IFN and ribavirin (RBV) treatments.To assess the effects of HCV amino acid variations in NS3, NS4A and NS4B of HCV subtypes 1a, 1b, 3a, 3b and 6f on the response to pegylated interferon (Peg-IFN) and RBV therapy.One hundred and thirty four HCV isolates of genotypes 1a, 1b, 3a, 3b and 6f obtained from HCV patients both before and at week 4 of treatments were evaluated. Amino acid sequences of NS3, NS4A and NS4B were analyzed and in compared to reference sequences of corresponding genotypes.The data revealed that amino acid variations within the full-length NS3, protease and helicase domains of NS3 of HCV 1a from responders were significantly higher than those from treatment failure groups as compared to reference sequences of each corresponding genotype. Similar results were observed in the full-length and helicase domain but not in the protease domain of HCV 1b. However, the number of amino acid variations in NS3 of HCV 3a, 3b and 6f as well as in NS4A and NS4B showed no difference between the viruses from responders and treatment failure group. Analysis of amino acid variations both before and at week 4 of treatment revealed that the mean number of amino acid variation in the full-length NS3 of HCV 3a and 3b from responders were also significantly higher than those from the treatment failure group.Our study suggests that the increase of amino acid variations within the NS3 protein of HCV 1a, 1b, 3a and 3b were associated with the response to Peg-IFN and RBV treatment in Thai patients.

Prediction of nonsynonymous single nucleotide polymorphisms in human disease-associated genes.

Analysis of human genetic variation can shed light on the problem of the genetic basis of complex disorders. Nonsynonymous single nucleotide polymorphisms (SNPs), which affect the amino acid sequence of proteins, are believed to be the most frequent type of variation associated with the respective disease phenotype. Complete enumeration of nonsynonymous SNPs in the candidate genes will enable further association studies on panels of affected and unaffected individuals. Experimental detection of SNPs requires implementation of expensive technologies and is still far from being routine. Alternatively, SNPs can be identified by computational analysis of a publicly available expressed sequence tag (EST) database following experimental verification. We performed in silico analysis of amino acid variation for 471 of proteins with a documented history of experimental variation studies and with confirmed association with human diseases. This allowed us to evaluate the level of completeness of the current knowledge of nonsynonymous SNPs in well studied, medically relevant genes and to estimate the proportion of new variants which can be added with the help of computer-aided mining in EST databases. Our results suggest that approx. 50% of frequent nonsynonymous variants are already stored in public databases. Computational methods based on the scan of an EST database can add significantly to the current knowledge, but they are greatly limited by the size of EST databases and the nonuniform coverage of genes by ESTs. Nevertheless, a considerable number of new candidate nonsynonymous SNPs in genes of medical interest were found by EST screening procedure.

Animal Colonies — Development and Function through Time

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.