Biology subjects

Cusco, A.

Publications and source records attributed to Cusco, A..

8 recordsLinked to original sources

bioRxiv2026

Long-read metagenomic sequencing reveals novel lineages and functional diversity in urban soil microbiome

City parks and other urban green spaces can bring significant benefits to the physical and mental health of city residents. However, there is limited knowledge about the microbial communities inhabiting these urban soils. Here, we applied long-read metagenomic sequencing to 58 urban soil samples from two major cities in China, enabling genome-resolved reconstruction of microbial diversity at unprecedented contiguity. We recovered 7,949 medium- and high-quality metagenome-assembled genomes, comprising 4,171 species-level genome bins, of which over 97% represent previously undescribed species. Long-read assemblies revealed extensive secondary metabolic capacity, including more than 30,000 biosynthetic gene clusters, which were highly contiguous compared with those from fragmented short-read assemblies. Beyond secondary metabolism, we uncovered over 2 million small protein families, including hundreds that are strongly enriched in the neighbourhood of defense systems and mobile genetic elements, highlighting their overlooked role in urban soils. These findings expand our understanding of the functional diversity of urban soil microbiomes and provide new insights with implications for urban public health.

bioinformatics↗

bioRxiv2025

Capturing global pet dog gut microbial diversity and hundreds of near-finished bacterial genomes by using long-read metagenomics in a Shanghai cohort

Pet dogs are considered part of the family, and understanding their gut microbiomes can provide insights into both animal and household health. Most comprehensive studies, however, relied on short-read sequencing, resulting in fragmented MAGs that miss mobile elements, antimicrobial-resistance genes, and ribosomal genes. Here, we applied deep long-read metagenomics (polished with short-reads) to fecal samples from 51 urban pet dogs in Shanghai, generating 2,676 MAGs--representing 320 bacterial species--, of which [~]72% achieved near-finished quality, often improving on the corresponding reference public genome. Comparisons with external datasets showed that our Shanghai-based MAG catalog is representative of pet dogs worldwide (median read mapping of >90%). Moreover, we recovered circular extrachromosomal elements, including those linked to antimicrobial resistance, which were also detected in external dog gut datasets. In conclusion, we provide a high-quality reference resource and demonstrate the power of deep long-read metagenomics to resolve microbial diversity in complex host-associated microbiomes.

microbiology↗

bioRxiv2021

Novel canine high-quality metagenome-assembled genomes, prophages, and host-associated plasmids by long-read metagenomics together with Hi-C proximity ligation

Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors. We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range. In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

microbiology↗

bioRxiv2020

Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples.

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to the species level and in real-time. We assess the taxonomy skills of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the Whats in my pot (WIMP) classifier. We used the ZymoBIOMICS mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp. We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

microbiology↗

bioRxiv2019

Applying Nanopore sequencing to a One-Health scenario for colistin resistance transmission among pigs, cows and the farmer

One-Health studies applying massive-parallel and single-molecule sequencing are a suitable approximation to try to understand how antibiotic resistances flow between the human-animal-environment scenario. Colistin has been withdrawn in human medicine due to its toxicity, limiting its usage as a last-resort treatment option for multidrug-resistant Gram-negative bacteria. However, it is still used orally to treat Enterobacteriaceae infections in veterinary medicine. Since 2015, colistin resistance appeared to be located in mobile genetic elements, raising the concern of the likelihood of transmission by horizontal gene transfer between animals and humans. In this study, 202 faecal samples were collected in a mixed farm from pigs, calves, and the farmer. PCR for the mcr-1 gene was positive for 18 of the isolates, and Nanopore sequencing allowed us to determine the location of the gene, either on the chromosome or in plasmids. Three types of replicons were found within the positive isolates harbouring the mcr-1: IncX4, IncI2, and IncHI2. Four different genetic contexts probably indicate different stages of gene stabilization, either in the chromosome or plasmid, with ISApl1 as the main insertion element flanking the gene. Moreover, 43 other resistance genes were found in our samples, related to more than six different antibiotic families (e.g. aminoglycosides, lincosamides, beta-lactams, macrolides, trimethoprim, phenicols, and sulphonamides). We found resistance genes against colistin and that six antibiotic families together in at least one of the isolates from human, swine, and bovine. Isolate 15B-22 harboured one plasmid with seven resistance genes related to four families of antibiotics other than polymyxins, meaning that there are more chances to maintain colistin resistance even with the withdrawn of colistin. Nanopore long reads allowed us to assemble the DNA elements within the isolates easily and determine the genetic context of the mcr-1 gene. Furthermore, they allowed us to describe and locate more antimicrobial resistance genes to other antibiotic families and antiseptic compounds.

microbiology↗

bioRxiv2019

Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

Long-read metagenomics -using single-molecule sequencers- has the potential to assembly entire genomes, even from complex metagenomics datasets. Using long-read metagenomics with Nanopore sequencing in pooled samples, we aim to improve the individual taxonomic profiles obtained with V4 16S rRNA massive sequencing and to assemble the fecal metagenome of healthy dogs.\n\nFecal samples from healthy dogs were sequenced individually using V4 16S rRNA gene and in pools using a shallow metagenomics approach with Nanopore sequencing. Long-read metagenomics allowed us refining the V4 16S rRNA results up to the species level and determining the main bacterial species inhabiting on fecal microbiota of our cohort of healthy dogs. Among them, the most abundant were Fusobacterium varium; Megamonas hypermegale; Bacteroides vulgatus; Blautia hansenii; Clostridium perfringens; and Clostridoides difficile. Once performed the metagenome assembly, one contig was suggested to be circular and hit to an uncultured crAssphage.\n\nTo conclude, shallow long-read metagenomics with pooled samples using MinION allowed characterizing the dog fecal microbiota at lower taxonomic level, such as bacterial species. The assembly of the reads retrieved a contig that represents a circular draft genome of an uncultured CrAssphage from dog fecal samples that is one of the most abundant bacteriophages in the human gut.

microbiology↗

bioRxiv2018

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

BackgroundProfiling microbiome on low biomass samples is challenging for metagenomics since these samples are prone to present DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA ([~]1,500 bp) and the whole rrn operon (16S rRNA gene - ITS - 23S rRNA gene; 4,500 bp).\n\nMethodsWe sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS) and two pools of lowbiomass samples (dog skin). Nanopore sequencing was performed on MinION (Oxford Nanopore Technologies) using 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using WIMP workflow on EPI2ME (ONT) or Minimap2 software with rrn database.\n\nResultsFull-length 16S rRNA and the rrn operon retrieved the microbiota composition from the bacterial isolate, the mock communities and the complex skin samples, even at the genus and species level. For Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in [~]98% of the cases with rrn operon as the marker, and [~]68% of the cases with 16S rRNA gene respectively. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances.\n\nConclusionsBoth full-length 16S rRNA and the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, rrn operon would be the best choice.

microbiology↗

bioRxiv2017

Using MinION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3rd generation sequencers.\n\nWe aimed to assess the performance of nanopore sequencing using MinION on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2nd generation sequencers.

microbiology↗