GPA: A Microbial Genetic Polymorphisms Assignments Tool in Metagenomic Analysis by Bayesian Estimation

Identifying antimicrobial resistant(AMR) bacteria in metagenomics samples is essential for public health and food safety. Next-generation sequencing(NGS) technology has provided a powerful tool in identifying the genetic variation and constructing the correlations between genotype and phenotype in humans and other species. However, for complex bacterial samples, there lacks a powerful bioinformatic tool to identify genetic polymorphisms or copy number variations(CNVs) for given genes. Here we provide a Bayesian framework for genotype estimation for mixtures of multiple bacteria, named as Genetic Polymorphisms Assignments(GPA). Simulation results showed that GPA has reduced the false discovery rate(FDR) and mean absolute error(MAE) in CNV and single nucleotide variant(SNV) identification. This framework was validated by whole-genome sequencing and Pool-seq data from Klebsiella pneumoniae with multiple bacteria mixture models, and showed the high accuracy in the allele fraction detections of CNVs and SNVs in AMR genes between two populations. The quantitative study on the changes of AMR genes fraction between two samples showed a good consistency with the AMR pattern observed in the individual strains. Also, the framework together with the genome annotation and population comparison tools has been integrated into an application, which could provide a complete solution for AMR gene identification and quantification in unculturable clinical samples. The GPA package is available at https://github.com/IID-DTH/GPA-package.     

Fungal communities associated with acorn woodpeckers and their excavations

Wood-decay fungi soften wood, putatively providing opportunities for woodpeckers to excavate an otherwise hard substrate, yet the fungal community composition in tree cavities and the specificity of these relationships is largely unknown. We used high-throughput amplicon sequencing of the fungal ITS2 region to examine the fungal communities associated with acorn woodpeckers (Melanerpes formicivorus) and their cavities in mature valley oak (Quercus lobata) and blue oak (Q. douglasii) trees in an oak savannah of central coastal California, USA. Acorn woodpeckers and their excavations harbored over 1500 fungal taxa, including more than 100 putative wood-decay fungi. The fungal communities found on the birds were more similar to those found in excavated cavities than those found in trees without excavated holes. These results suggest that symbiotic associations between acorn woodpeckers and fungi are highly diverse, with low specificity. Symbiotic associations between cavity-excavators and fungi are likely more common and widespread than previously thought.     

Differential expression profiles of miRNA in the serum of sarcopenic rats

As the geriatric population and life expectancy increase, the interest in preventing geriatric diseases, such as sarcopenia, is increasing. However, the causes of sarcopenia are unclear, and current diagnostic methods for sarcopenia are unreliable. We hypothesized that the changes in the expression of certain miRNAs may be associated with the pathophysiology of sarcopenia. Herein, we analyzed the miRNA expression profiles in the blood of young (3-months-old) healthy rats, old sarcopenic (17-months-old) rats, and age-matched (17-months-old) control rats. The changes in miRNA expression levels were analyzed using Bowtie 2 software. A total of 523 miRNAs were detected in the rat serum. Using scatter plots and clustering heatmap data, we found 130 miRNAs that were differentially expressed in sarcopenic rats (>2-fold change) compared to the expression in young healthy and age-matched control rats. With a threshold of >5-fold change, we identified 14 upregulated miRNAs, including rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-133c, rno-miR-208a-3p, and rno-miR434-5p among others in the serum of sarcopenic rats. A protein network map based on these 14 miRNAs identified the genes involved in skeletal muscle differentiation, among which Notch1, Egr2, and Myocd represented major nodes. The data obtained in this study are potentially useful for the early diagnosis of sarcopenia and for the identification of novel therapeutic targets for the treatment and/or prevention of sarcopenia.     

Quality control and integration of genotypes from two calling pipelines for whole genome sequence data in the Alzheimer's disease sequencing project

The Alzheimer's Disease Sequencing Project (ADSP) performed whole genome sequencing (WGS) of 584 subjects from 111 multiplex families at three sequencing centers. Genotype calling of single nucleotide variants (SNVs) and insertion-deletion variants (indels) was performed centrally using GATK-HaplotypeCaller and Atlas V2. The ADSP Quality Control (QC) Working Group applied QC protocols to project-level variant call format files (VCFs) from each pipeline, and developed and implemented a novel protocol, termed “consensus calling,” to combine genotype calls from both pipelines into a single high-quality set. QC was applied to autosomal bi-allelic SNVs and indels, and included pipeline-recommended QC filters, variant-level QC, and sample-level QC. Low-quality variants or genotypes were excluded, and sample outliers were noted. Quality was assessed by examining Mendelian inconsistencies (MIs) among 67 parent-offspring pairs, and MIs were used to establish additional genotype-specific filters for GATK calls. After QC, 578 subjects remained. Pipeline-specific QC excluded ~12.0% of GATK and 14.5% of Atlas SNVs. Between pipelines, ~91% of SNV genotypes across all QCed variants were concordant; 4.23% and 4.56% of genotypes were exclusive to Atlas or GATK, respectively; the remaining ~0.01% of discordant genotypes were excluded. For indels, variant-level QC excluded ~36.8% of GATK and 35.3% of Atlas indels. Between pipelines, ~55.6% of indel genotypes were concordant; while 10.3% and 28.3% were exclusive to Atlas or GATK, respectively; and ~0.29% of discordant genotypes were. The final WGS consensus dataset contains 27,896,774 SNVs and 3,133,926 indels and is publicly available.     

宏基因组的生物信息分析

基于高通量测序的宏基因组学研究是近年来的研究热点之一。宏基因组的生物信息分析正在逐渐完善成熟．各种分析软件和流程的开发与应用,极大地促进了宏基因组研究的发展,特别是在遗传与进化、基因发现、宏基因组和人类疾病的相关研究等方面取得了显著成果。本文旨在结合宏基因组学的研究内容和研究方向,对宏基因组的生物信息分析方法进行综述,探讨宏基因组的生物信息分析面临的机遇和挑战。     

Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.     

Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.     

一株猴泡沫病毒野毒Pol基因的cDNA的直接测序

A 377 Kunming cell line of Rhesus Monkey Kidney (RMK) from Centre for Medical Pr imates was amplified the cDNA fragments of the pol gene by Nested-PCR,which of a natured infected SFV showed typical CPE after cultured for three weeks,sequence s of products position(nt 5989-6343)were determined directly.The result indicate d that the nucleotid sequences in the region had 48 nucleotids difference(10.78% ) between SFV infected cell and SFV-1 in GENE BANK,and there was 45 nucleotides difference(10.11%) compared with SFVmac in the region, the test with VspⅠ showe d aligment.     

A Reexamination of the Phylogenetic Position of Callimico (Primates) Incorporating New Mitochondrial DNA Sequence Data

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA^His, tRNA^Ser, and tRNA^Leu genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico. Received: 20 March 1997 / Accepted: 17 December 1997