Illumina MiSeq 16S rRNA Gene Library Preparation for Poultry Processing Microbiome Analyses.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Dynamics of drinking water biofilm formation associated with Legionella spp. colonization.

Understanding how Legionella spp. proliferate in multispecies biofilms is essential to develop strategies to control their presence in building plumbing. Here, we analyzed biofilm formation and Legionella spp. colonization on new plumbing material during 8 weeks. Biofilm formation was characterized by an initial increase in intact cell concentrations up to 9.5 × 105 cells/cm2, followed by a steady decrease. We identified Comamonas, Caulobacter, Schlegella, Blastomonas and Methyloversatilis as pioneer genera in the biofilm formation process. Importantly, L. pneumophila was the dominant Legionella spp. and rapidly colonized the biofilms, with culturable cell concentrations peaking at 3.1 × 104 MPN/cm2 after 4 weeks already. Moreover, several Legionella species co-occurred and had distinct dynamics of biofilm colonization. Vermamoeba vermiformis (V. vermiformis) was the dominant protist identified with 18S rRNA gene amplicon sequencing. Together our results highlight that biofilm formation upon introduction of new building plumbing material is a dynamic process where pathogenic Legionella species can be part of the earliest colonizers.

Genus-targeted markers for the taxonomic identification and monitoring of coagulase-positive and coagulase-negative Staphylococcus species.

The Staphylococcus genus comprises multiple pathogenic and opportunistic species that represent a risk to public health. Epidemiological studies require accurate taxonomic classification of isolates with enough resolution to distinguish clonal complexes. Unfortunately, 16 S rRNA molecular analysis and phenotypic characterization cannot distinguish all species and do not offer enough resolution to assess intraspecific diversity. Other approaches, such as Multilocus Sequence Tagging, provide higher resolution; however, they have been developed for Staphylococcus aureus and a few other species. Here, we developed a set of genus-targeted primers using five orthologous genes (pta, tuf, tpi, groEs, and sarA) to identify all Staphylococcus species within the genus. The primers were initially evaluated using 20 strains from the Collection of Microorganisms of Interest in Animal Health from AGROSAVIA (CMISA), and their amplified sequences were compared to a set of 33 Staphylococcus species. This allowed the taxonomic identification of the strains even on close species and the establishment of intraspecies diversity. To enhance the scope and cost-effectiveness of the proposed strategy, we customized the primer sets for an Illumina paired-end amplicon protocol, enabling gene multiplexing. We assessed five genes across 177 strains, generating 880 paired-end libraries from the CMISA. This approach significantly reduced sequencing costs, as all libraries can be efficiently sequenced in a single MiSeq run at a fraction (one-fourth or less) of the cost associated with Sanger sequencing. In summary, this method can be used for precise identification and diversity analysis of Staphylococcus species, offering an advancement over traditional techniques in both resolution and cost-effectiveness.

Genomic analysis of an acute gastroenteritis outbreak caused by rotavirus C in Hebei, China.

Rotavirus group C is an important cause of sporadic cases and outbreaks of gastroenteritis worldwide. Whole-Genome sequences of human rotavirus C (RVC) in public databases are limited. We performed genome sequencing to analyze a RVC outbreak of acute gastroenteritis in China. Samples from 22 patients were screened for pathogens using RT-PCR, and six samples were positive for rotavirus. Whole-Genome sequencing analysis showed that the outbreak strain SJZ217 belongs to the G4-P[2]-I2-R2-C2-M3-A2-N2-T2-E2-H2 genotype and shares almost identical genomic sequences with Chungnam isolated in Korea. Phylogenetic analysis revealed strain SJZ217 also fell into a cluster with rotavirus C strains from Japan and Europe. Reassortment in the VP4 fragment was observed. These results helped to understand the genetic diversity and possible spread of RVC strains.

Prokaryotic communities profiling of Indonesian hot springs using long-read Oxford Nanopore sequencing.

OBJECTIVES: Indonesia's location at the convergence of multiple tectonic plates results in a unique geomorphological feature with abundant hot springs. This study pioneers the metagenomic exploration of Indonesian hot springs, harbouring unique life forms despite high temperatures. The microbial community of hot springs is taxonomically versatile and biotechnologically valuable. 16s rRNA amplicon sequencing of the metagenome is a viable option for the microbiome investigation. This study utilized Oxford Nanopore's long-read 16 S rRNA sequencing for enhanced species identification, improved detection of rare members, and a more detailed community composition profile. DATA DESCRIPTION: Water samples were taken from three hot springs of the Bali, Indonesia (i) Angseri, 8.362503 S, 115.133452 E; (ii) Banjar, 8.210270 S, 114.967063 E; and (iii) Batur, 8.228806 S, 115.404829 E. BioLit Genomic DNA Extraction Kit (SRL, Mumbai, India) was used to isolate DNA from water samples. The quantity and quality of the DNA were determined using a NanoDrop™ spectrophotometer and a Qubit fluorometer (Thermo Fisher Scientific, USA). The library was created using Oxford Nanopore Technology kits, and the sequencing was done using Oxford Nanopore's GridION platform. All sequencing data was obtained in FASTQ files and filtered using NanoFilt software. This dataset is valuable for searching novel bacteria diversity and their existence.

Campylobacter californiensis sp. nov., isolated from cattle and feral swine.

Nine Campylobacter strains were isolated from cattle and feral swine faeces: three were recovered during a 2007 Campylobacter-associated outbreak linked to a dairy, and the other six were isolated during a 2009-2010 survey of farms and ranches in Central California. The species identification of these strains could not be determined by 16S rRNA gene sequencing but were most similar to Campylobacter concisus and Campylobacter mucosalis. Additional atpA typing indicated that the nine strains composed a discrete novel clade related to C. concisus and C. mucosalis. A polyphasic study was undertaken here to clarify their taxonomic position. Phylogenetic analyses were performed based on 16S rRNA gene sequences and the concatenated sequences of 330 core genes. The core gene analysis placed the nine strains into a clade well separated from the other Campylobacter taxa, indicating that these strains represent a novel Campylobacter species. Pairwise digital DNA-DNA hybridization and average nucleotide identity values between these strains and other campylobacters are lower than 16 and 73%, respectively, further supporting their placement into a novel taxon. Standard phenotypic testing was performed. All strains are microaerobic or anaerobic, motile, Gram-negative, slightly-curved rods that are oxidase positive but catalase negative. Strains can be distinguished from the other catalase-negative Campylobacter species using phenotypic markers such as motility, oxidase activity, cephalothin resistance, hippuricase activity, growth at 30 °C, and α-haemolysis. The data presented here show that these strains represent a novel species within Campylobacter, for which the name Campylobacter californiensis sp. nov. (type strain RM6914T=LMG 32304T=CCUG 75329T) is proposed.

HyLight: Strain aware assembly of low coverage metagenomes.

Different strains of identical species can vary substantially in terms of their spectrum of biomedically relevant phenotypes. Reconstructing the genomes of microbial communities at the level of their strains poses significant challenges, because sequencing errors can obscure strain-specific variants. Next-generation sequencing (NGS) reads are too short to resolve complex genomic regions. Third-generation sequencing (TGS) reads, although longer, are prone to higher error rates or substantially more expensive. Limiting TGS coverage to reduce costs compromises the accuracy of the assemblies. This explains why prior approaches agree on losses in strain awareness, accuracy, tendentially excessive costs, or combinations thereof. We introduce HyLight, a metagenome assembly approach that addresses these challenges by implementing the complementary strengths of TGS and NGS data. HyLight employs strain-resolved overlap graphs (OG) to accurately reconstruct individual strains within microbial communities. Our experiments demonstrate that HyLight produces strain-aware and contiguous assemblies at minimal error content, while significantly reducing costs because utilizing low-coverage TGS data. HyLight achieves an average improvement of 19.05% in preserving strain identity and demonstrates near-complete strain awareness across diverse datasets. In summary, HyLight offers considerable advances in metagenome assembly, insofar as it delivers significantly enhanced strain awareness, contiguity, and accuracy without the typical compromises observed in existing approaches.

Identification of three new HLA-A intronic variants by next-generation sequencing.

Three novel HLA-A intronic variants, HLA-A*03:01:01:121, HLA-A*29:02:01:41 and HLA-A*30:02:01:24, detected by next-generation sequencing.

Characterisation of the novel HLA-C*01:262 allele by sequencing-based typing.

HLA-C*01:262 differs from HLA-C*01:02:01:01 by one nucleotide substitution in codon 150 in exon 3.

Detection of the HLA-B*40:78 allele in a Taiwanese individual.

One nucleotide substitution in codon 136 of HLA-B*40:02:01:01 results in a novel allele, HLA-B*40:78.

Yamadazyma thunbergiae sp. nov., a novel yeast species associated with Bengal clock vines and soil in Okinawa, Japan.

Two strains, designated JCM 36746T and JCM 36749, were isolated from Bengal clock vine (Thunbergia grandiflora) and soil, respectively, in Okinawa, Japan. Analysis of the internal transcribed spacer (ITS) regions and D1/D2 domains of the large subunit rRNA gene sequences revealed identical sequences in both strains, indicating that they belong to the same species. Sequence analysis and physiological characterization identified these strains as representing a novel yeast species in the genus Yamadazyma. The sequence similarities of the concatenated ITS regions and D1/D2 domains indicated that JCM 36746T and JCM 36749 formed a well-supported distinct from closely related species belonging to the Yamadazyma clade, including Candida dendronema, C. diddensiae, C. germanica, C. kanchanaburiensis, C. naeodendra, C. vaughaniae, Y. akitaensis, Y. koratensis, Y. nakazawae, Y. philogaea, Y. phyllophila, Y. siamensis, Y. ubonensis, and three undescribed species, comprising Candida aff. naeodendra/diddensiae Y151, Candida sp. GE19S08, and Yamadazyma sp. strain NYNU 22830. The sequences of the D1/D2 domains and ITS regions differed in nucleotide substitutions by 1.51% and 2.57% or greater, respectively, from those of the previously described and undescribed related species. In addition, the physiological characteristics of the novel species were distinct from those of the closely related described species. On the basis of these findings, we propose the name Yamadazyma thunbergiae sp. nov. to classify this species within the genus Yamadazyma. The holotype used is JCM 36746T (ex-type strains CBS 18614 and NBRC 116657). The MycoBank accession number is MB 853823.

Pseudoalteromonas simplex sp. nov. Isolated from the Skin of Bandtail Puffer Fish (Sphoeroides spengleri).

A novel bacterial isolate A520T (A520T = CBAS 737T = CAIM 1944T) was obtained from the skin of bandtail puffer fish Sphoeroides spengleri (Tetraodontidae Family), collected in Arraial do Cabo (Rio de Janeiro, Brazil). A520T is Gram-stain-negative, flagellated and aerobic bacteria. Optimum growth occurs at 25-30 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.5 Mb (4082 coding genes and G+C content of 41.1%). The closest phylogenetic neighbor was Pseudoalteromonas shioyasakiensis JCM 18891T (97.9% 16S rRNA sequence similarity, 94.8% Average Amino Acid Identity, 93% Average Nucleotide Identity and 51.8% similarity in Genome-to-Genome-Distance). Several in silico phenotypic features are useful to differentiate A520T from its closest phylogenetic neighbors, including trehalose, D-mannose, cellobiose, pyrrolidonyl-beta-naphthylamide, starch hydrolysis, D-xylose, lactose, tartrate utilization, sucrose, citrate, glycerol, mucate and acetate utilization, malonate, glucose oxidizer, gas from glucose, nitrite to gas, L-rhamnose, ornithine decarboxylase, lysine decarboxylase and yellow pigment. The genome of the novel species contains 3 gene clusters (~ 66.81 Kbp in total) coding for different types of bioactive compounds that could indicate ecological roles pertaining to the bandtail puffer fish host. Based on genome-based taxonomic approach, strain A520T (A520T = CBAS 737T = CAIM 1944T) is proposed as a new species, Pseudoalteromonas simplex sp. nov.

Chromobacterium indicum sp. nov., a Pigment-Producing Bacterium Isolated from Soil.

A purple colony, designated as TRC1.1.SA was isolated from a tea garden soil sample. It was a Gram-negative, rod-shaped, non-spore-forming and motile bacterium. The strain TRC1.1.SAT grew aerobically at temperatures 15-37 â„ƒ and pH levels 5.0-9.0. It showed both oxidase and catalase activity. The 16S rRNA gene sequence blast analysis revealed identity with the members of the genus Chromobacterium. The maximum identity was with the type strains of species Chromobacterium piscinae CCM 3329T (99.8%), C. vaccinii MWU205T (99.7%), and C. violaceum ATCC 12472T (98.7%). However, the average nucleotide identity (ANI) of the genome sequence showed less than 96% similarity with all species of the genus Chromobacterium. Further, digital DNA-DNA hybridization (dDDH) revealed the highest identity of 63.4% with its phylogenetic relative C. piscinae CCM 3329T. The G + C content of the strain was 63.9%. The major polar lipids identified were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphoglyceraldehyde (PG). Fatty acid analysis showed C16:0, C16:1ω7c, C17:0 cyclo, and C18:1ω7c as the major fatty acids. RAST and antiSMASH analyses of the genome revealed the presence of a biosynthetic gene cluster (BGC) involved in the production of violacein pigment, as observed for type species C. violaceum ATCC 12472T. Considering the phenotypic differences and genomic identity, strain TRC1.1.SAT is assigned as a novel species of the genus Chromobacterium, for which the name Chromobacterium indicum is proposed. The type strain of prospective species is designated as TRC1.1.SAT (= MTCC 13391T; JCM 36723T; = KCTC 8324T).

Complete genome sequence analysis and Pks genes identification of Brevibacillus brevis FJAT-0809-GLX with a broad inhibitory spectrum against phytopathogens.

Brevibacillus brevis FJAT-0809-GLX has a broad spectrum of antimicrobial activity. Understanding the molecular basis of biocontrol ability of B. brevis will allow us to develop effective microbial agents for sustainable agriculture. In this study, we present the complete and annotated genome sequence of FJAT-0809-GLX. The complete genome size of B. brevis FJAT-0809-GLX was 6,137,019 bp, with 5688 predicted coding sequences (CDS). The average GC content of 47.38%, and there were 44 copies of the rRNAs operon (16S, 23S and 5S RNA), and 127 tRNA genes. A total of 11,162 genes were functionally annotated with the COG, GO, and KEGG databases, and 123 genes belonged to CAZymes. Genomic secondary metabolite analysis indicated 13 clusters encoding potential new antimicrobials. FJAT-0809-GLX was designated as B. brevis according to average nucleotide polymorphism (ANI) and phylogenetic analysis. The pangenome consisted of 7141 homologous genes, and 4469 homologous genes shared by B. brevis FJAT-0809-GLX, B. brevis NBRC100599, B. brevis DSM30, and B. brevis NCTC2611. The number of unique homologous genes of B. brevis FJAT-0809-GLX (419 genes) and B. brevis NBRC100599 (480 genes) were much more than those in B. brevis DSM30 (13 genes), and B. brevis NCTC2611 (6 genes). Nine gene clusters encoding for secondary metabolite biosynthesis were compared in the genome of B. brevis FJAT-0809-GLX with those of B. brevis NBRC100599, B. brevis DSM30 and B. brevis NCTC2611, and the gene clusters encoding for lantipeptide and transatpks-otherks only existed in genome of B. brevis FJAT-0809-GLX. The 11 BbPks genes were included in the B. brevis FJAT-0809-GLX genome, which contained the conserved PS-DH domain. The relative expression of BbPksL, BbPksM2, BbPksM3, BbPksN3, BbPksN4 and BbPksN5 reached a maximum at 120 h and then decreased at 144 h. Our results provided detailed genomic and Pks genes information for the FJAT-0809-GLX strain, and lid a foundation for studying its biocontrol mechanisms.

Adlercreutzia wanghongyangiae sp. nov., and Adlercreutzia shanghongiae sp. nov., two new members of the genus Adlercreutzia isolated from plateau pika (Ochotona curzoniae).

Four anaerobic, Gram-stain-positive, non-motile, non-sporulating rod-shaped bacterial strains (R7T, R21, R22 and R25T) were isolated from the intestinal contents of plateau pika (Ochotona curzoniae) collected from the Qinghai-Tibet Plateau, PR China. The four isolates grew at between 25 and 42 °C (optimally at 35-37 °C), and with 0.3-3.3% NaCl (w/v) [optimum, 1.3% (w/v)]. Adding l-arginine to the medium could promote their growth. Strains R7T and R21 were most closely related to Adlercreutzia caecimuris B7T (97.48% 16S rRNA gene sequence similarity). Strains R25T and R22 were most closely related to Adlercreutzia equolifaciens DSM 19450T (98.25% 16S rRNA gene sequence similarity). The genome sequences of R7T and R25T were 2.89 and 2.90 Mb in size with 63.6 and 62.8 mol% DNA G+C contents, respectively. Phylogenetic analysis based on 16S rRNA gene sequences and core genes revealed that R7T and R21 were most closely related to A. caecimuris B7T and Adlercreutzia mucosicola DSM 19490T, whereas R25T and R22 were most closely related to A. equolifaciens DSM 19450T and Adlercreutzia rubneri ResAG-91T. R7T, R25T and the closely related species had average nucleotide identity (ANI) values of 81.9-83.2% as well as digital DNA-DNA hybridisation (dDDH) values between 27.3 and 27.9%, which clearly indicated that they represent two novel species within the genus Adlercreutzia. For R7T and R25T, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the whole cell sugars included galactose, glucose and ribose. On the basis of these results, we propose that strains R7T and R25T represent two novel species of the genus Adlercreutzia, namely Adlercreutzia wanghongyangiae sp. nov. and Adlercreutzia shanghongiae sp. nov., respectively. The type strains are R7T (=GDMCC 1.4459T=KCTC 25860T) and R25T (=GDMCC 1.4458T=KCTC 25861T).

Characterization of the Novel HLA-C*07:01:126 Allele by Sequencing-Based Typing.

HLA-C*07:01:126 differs from HLA-C*07:01:01:01 by one nucleotide substitution in codon 328 in exon 7.

The HLA-C*14:152 Allele Was Identified Using a Next-Generation Sequencing Method.

HLA-C*14:152 differs from HLA-C*14:02:01:01 by a non-synonymous nucleotide substitution in exon 5.

Characterization of the Novel HLA-A*24:02:169 Allele by Sequencing-Based Typing.

HLA-A*24:02:169 differs from HLA-A*24:02:01:01 by one nucleotide substitution in codon -23 in exon 1.