Molecular identification of genus Pipistrellus (Mammalia: Chiroptera) from Fata region, Pakistan

A total of 10 specimens were captured from selected sites of Bajaur Agency FATA, Pakistan using mist nets. The captured specimens were morphologically identified and various morphometric measurements were taken. The head and Body length (HB) of Pipistrellus coromondra and Pipistrellus kuhlii lepidus (n=10) was 43±0.11 mm and 45±1.1 respectively. Morphologically identified Pipistrellus kuhlii confirmed as Pipistrellus kuhlii lepidus based on 16S rRNA sequences. The DNA sequences were submitted to GenBank and accession numbers were obtained (MN 719478 and MT430902). The available 16S rRNA gene sequences of Pipistrellus coromondra and Pipistrellus kuhlii lepidus were retrieved from NCBI and incorporated in N-J tree analysis. Overall, the interspecific genetic variations among Pipistrellus coromondra and Pipistrellus kuhlii lepidus were 8% and 1% respectively. In our recommendation, a comprehensive molecular identification of bats is need of hour to report more cryptic and new species from Pakistan.

The electronic tree of life (eTOL): a net of long probes to characterize the human microbiome from RNA-seq data

Background Microbiome analysis generally requires PCR-based or metagenomic shotgun sequencing, sophisticated programs, and large volumes of data. Alternative approaches based on widely available RNA-seq data are constrained because of sequence similarities between the transcriptomes of microbes/viruses and those of the host, compounded by the extreme abundance of host sequences in such libraries. Current approaches are also limited to specific microbial groups. There is a need for alternative methods of microbiome analysis that encompass the entire tree of life. Results We report a method to specifically retrieve non-human sequences in human tissue RNA-seq data. For cellular microbes we used a bioinformatic 'net', based on filtered 64-mer small subunit rRNA sequences across the Tree of Life (the 'electronic tree of life', eTOL), to comprehensively (98%) entrap all non-human rRNA sequences present in the target tissue. Using brain as a model, retrieval of matching reads, re-exclusion of human-related sequences, followed by contig building and species identification, is followed by reconfirmation of the abundance and identity of the corresponding species groups. We provide methods to automate this analysis. A variant approach is necessary for viruses. Again, because of significant matches between viral and human sequences, a 'stripping' approach is essential. In addition, contamination during workup is a potential problem, and we discuss strategies to circumvent this issue. To illustrate the versatility of the method, we report the use of the eTOL methodology to unambiguously identify exogenous microbial and viral sequences in human tissue RNA-seq data across the entire tree of life including Archaea, Bacteria, Chloroplastida, basal Eukaryota, Fungi, and Holozoa/Metazoa, and discuss the technical and bioinformatic challenges involved. Conclusions This generic methodology may find wider application in microbiome analysis including diagnostics.

Genetic diversity of oral streptococci in the guinea pig as assessed by sequence analysis of the 16S rRNA and groEL genes

The aim of the present study was to characterize bacteria of the genus Streptococcus isolated from the oral cavity of the guinea pig as well as to assess the significance of these microorganisms as potential veterinary and human pathogens. Sixty-two streptococcal isolates recovered from 27 clinically healthy guinea pigs were examined genotypically by sequencing the 16S rRNA and groEL genes. Among these isolates, only 13 could be assigned to a species described previously (mainly Streptococcus parasanguinis, S. mitis and S. suis), and the majority of the remaining ones differed considerably from the streptococcal species known to date (16S rRNA and groEL sequence similarities were < 97% and < 87%, respectively). Based on 16S rRNA sequences, these unidentified isolates were divided into seven groups (clades), of which clades I through III comprised most of the isolates examined and had also the widest distribution among guinea pig colonies. Upon groEL gene sequence analysis, however, members of the three clades grouped together without forming such distinct clusters. The remaining clades distinguished by 16S rRNA sequencing could also be discerned by the second gene, and they contained only a few isolates often restricted to one or a few animal colonies. The present work reveals that the guinea pig mouth is inhabited by a vast number of phylogenetically diverse, so far unrecognized populations of streptococci, most of them being apparently host-specific genomospecies. On the contrary, S. parasanguinis and S. mitis are also common human commensals and S. suis is a well-recognized zoonotic pathogen.

Genomic and Metabolic Features of an Unexpectedly Predominant, Thermophilic, Assistant Starter Microorganism, Thermus thermophilus, in Chinese Inner Mongolian Cheese

Inner Mongolian cheese is a traditional dairy product in China. It is produced without rennet, using naturally acidified milk that is simmered to achieve whey separation. In order to analyse the impact of simmering on the microbial community structure, high-throughput sequencing was performed to obtain bacterial 16S rRNA sequences from cheeses from the Ordos (ES), Ulanqab (WS), Horqin (KS) and Xilingol (XS) grasslands of Inner Mongolia. The relative abundance of an unexpected microorganism, Thermus thermophilus, ranged from 2% to 9%, which meant that its dominance was second only to that of lactic acid bacteria (LABs). Genome sequencing and fermentation validation were performed in T. thermophilus N-1 isolated from the Ordos, and it was determined that T. thermophilus N-1 could ingest and metabolise lactose in milk to produce lactate during the simmering process. T. thermophilus N-1 could also produce acetate, propionate, citrate and other organic acids through a unique acetate production pathway and a complete propionate production pathway and TCA cycle, which may affect texture and flavour development in Inner Mongolian cheese. Simultaneously, the large amount of citrate produced by T. thermophilus N-1 provides a necessary carbon source for continuous fermentation by LABs after the simmering step. Therefore, T. thermophilus N-1 contributes to cheese fermentation as a predominant, thermophilic, assistant starter microorganism unique to Chinese Inner Mongolian cheese.

Simple Matching Using QIIME 2 and RDP Reveals Misidentified Sequences and an Underrepresentation of Fungi in Reference Datasets

Simple nucleotide matching identification methods are not as accurate as once thought at identifying environmental fungal sequences. This is largely because of incorrect naming and the underrepresentation of various fungal groups in reference datasets. Here, we explore these issues by examining an environmental metabarcoding dataset of partial large subunit rRNA sequences of Basidiomycota and basal fungi. We employed the simple matching method using the QIIME 2 classifier and the RDP Classifier in conjunction with the latest releases of the SILVA (138.1, 2020) and RDP (11, 2014) reference datasets and then compared the results with a manual phylogenetic binning approach. Of the 71 query sequences tested, 21 and 42% were misidentified using QIIME 2 and the RDP Classifier, respectively. Of these simple matching misidentifications, more than half resulted from the underrepresentation of various groups of fungi in the SILVA and RDP reference datasets. More comprehensive reference datasets with fewer misidentified sequences will increase the accuracy of simple matching identifications. However, we argue that the phylogenetic binning approach is a better alternative to simple matching since, in addition to better accuracy, it provides evolutionary information about query sequences.

Morphological and molecular analyses of parasitic barnacles (Crustacea: Cirripedia: Rhizocephala) in Korea: preliminary data for the taxonomy and host ranges of Korean species

Morphological and molecular analyses of Korean rhizocephalan barnacle species were performed to examine their host ranges and taxonomy. Morphological examination and molecular analysis of mtDNA cox1, 16S, and nuclear 18S rRNA sequences revealed nine rhizocephalan species from three genera of the two families, Sacculinidae and Polyascidae. Phylogenetic analysis of molecular sequences revealed two new species candidates in the genus Parasacculina, and three Sacculina species (S. pilosella, S. pinnotherae, and S. imberbis) were transferred to the genus Parasacculina. Examination of host ranges revealed higher host specificity and lower infestation rates in Korean rhizocephalan species than rhizocephalans from other geographic regions. This is the first report of the taxonomy, species diversity, and host ranges of Korean parasitic rhizocephalan barnacles based on their morphological and molecular analyses. More information from extensive sampling of parasitic barnacles from a wide range of crustacean host species is necessary to fully understand their taxonomy, prevalence on decapod hosts, and phylogenetic relationships among major rhizocephalan taxa.

ORPER: A Workflow for Constrained SSU rRNA Phylogenies

The continuous increase in sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects ever more complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow “ORPER”, for “ORganism PlacER”, containerized in Singularity, which allows the determination the phylogenetic position of a collection of organisms in the genomic landscape. ORPER constrains the phylogenetic placement of SSU (16S) rRNA sequences in a multilocus reference tree based on ribosomal protein genes extracted from public genomes. We demonstrate the utility of ORPER on the Cyanobacteria phylum, by placing 152 strains of the BCCM/ULC collection.

ORPER: a workflow for constrained SSU rRNA phylogenies

The continuous increase of sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects evermore complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow ORPER (containerized in Singularity), which allows determining the phylogenetic position of a collection of organisms in the genomic landscape. ORPER constrains the phylogenetic placement of SSU (16S) rRNA sequences in a multilocus reference tree based on ribosomal protein genes extracted from public genomes. We demonstrate the utility of ORPER on the Cyanobacteria phylum, by placing 152 strains of the BCCM/ULC collection.

Molecular genetic diversity of some rabbit breeds based on mitochondrial 16S rRNA sequences

<p>The present study was performed to assess the genetic variations among six rabbit breeds in Egypt based on mitochondrial 16S rRNA sequences. The length of partial mitochondrial 16S rRNA in the six rabbit breeds ranged from 546 bp to 558 bp. The sequenced regions were submitted to GenBank/NCBI under accession numbers (MW052052 - MW052057). The average content of A+T was 57% in all breeds. Among breeds, the percentages of genetic distance values were ranged from 0.000 to 0.004. The highest P-distance (0.004) was found between the New Zealand White breed and all other breeds. The results support the suitability of mitochondrial 16S rRNA for genetic diversity analysis of rabbit breeds and the applicability for future research on genetic relationships and the phylogeny of rabbit breeds.</p>

Diversity In Intestinal Microorganisms Contributes To The Bodysize Difference In Chinese Horses And Ponies

Background: Intestinal microbiota communities can reflect the digestion and metabolism of the host, as well as the appearance of the host. In China, there are various excellent horse and pony breeds with rich diversity in wither height. However, little is known about the community structure of the intestinal microbiota in horses, let alone the profound effects it causes. Results: Here in, we generated 16S rRNA sequences of intestinal microorganisms from 118 Chinese horses including Guanzhong horse, Debao pony, and Ningqiang pony. We found that the intestinal microbiota of horses is full of diversity, and Firmicutes, Bacteroidetes, and Spirochaetes, which is consistent with the special structure of the horse digestive tract. Interestingly, the abundance of Firmicutes and Bacteroidetes at the phylum level, showed a strong correlation with horse height, with R values of 0.82 and -0.86 respectively. Moreover, at the genus level, Coprococcus, Streptococcus, Treponema, and Prevotella demonstrated higher significance in terms of height, the prediction of PICRUSt2 function and multiple analyses of the metabolic pathways, and additionally, the metabolic pathways of energy intake and utilization were significantly enriched in horses relative to ponies (P<0.01). Notably, flora colonization in mouse littermates contributed to their broad development compared to the control group. Conclusions: Compared with ponies, the intestinal microbiota enabled better cellulose decomposition and energy uptake in horses; Thus horses could get more energy from food to meet their higher demand for larger body development than ponies. Therefore, our study helps to understand the gut microbiota patterns across equine breeds, which has the potential to advance approaches aimed at personalized microbial modifications in horse breeding.