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2022 ◽  
Vol 10 (1) ◽  
pp. 189
Author(s):  
Ignacio Vasquez ◽  
Ahmed Hossain ◽  
Hajarooba Gnanagobal ◽  
Katherinne Valderrama ◽  
Briony Campbell ◽  
...  

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55 ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies’ taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.


2022 ◽  
Author(s):  
jason.nguyen not provided ◽  
Tracy Lee ◽  
Rebecca Hickman ◽  
Natalie Prystajecky ◽  
John Tyson

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw


2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Xin Hua ◽  
Wei Song ◽  
Kangzong Wang ◽  
Xue Yin ◽  
Changqi Hao ◽  
...  

AbstractThe genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.


2022 ◽  
Author(s):  
Kar-Tong Tan ◽  
Michael Slevin ◽  
Matthew Meyerson ◽  
Heng Li

Nanopore long-read genome sequencing is emerging as a potential approach for the study of genomes including long repetitive elements like telomeres. Here, we report extensive basecalling induced errors at telomere repeats across nanopore datasets, sequencing platforms, basecallers, and basecalling models. We found that telomeres which are represented by (TTAGGG)n and (CCCTAA)n repeats in many organisms were frequently miscalled (~40-50% of reads) as (TTAAAA)n, or as (CTTCTT)n and (CCCTGG)n repeats respectively in a strand-specific manner during nanopore sequencing. We showed that this miscalling is likely caused by the high similarity of current profiles between telomeric repeats and these repeat artefacts, leading to mis-assignment of electrical current profiles during basecalling. We further demonstrated that tuning of nanopore basecalling models, and selective application of the tuned models to telomeric reads led to improved recovery and analysis of telomeric regions, with little detected negative impact on basecalling of other genomic regions. Our study thus highlights the importance of verifying nanopore basecalls in long, repetitive, and poorly defined regions of the genome, and showcases how such artefacts in regions like telomeres can potentially be resolved by improvements in nanopore basecalling models.


BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Nadja Brait ◽  
Büşra Külekçi ◽  
Irene Goerzer

Abstract Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.


2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Xuemin Dong ◽  
Shanshan Dong ◽  
Shengkai Pan ◽  
Xiangjiang Zhan

Abstract Background Understanding the transcriptome has become an essential step towards the full interpretation of the biological function of a cell, a tissue or even an organ. Many tools are available for either processing, analysing transcriptome data, or visualizing analysis results. However, most existing tools are limited to data from a single sequencing platform and only several of them could handle more than one analysis module, which are far from enough to meet the requirements of users, especially those without advanced programming skills. Hence, we still lack an open-source toolkit that enables both bioinformatician and non-bioinformatician users to process and analyze the large transcriptome data from different sequencing platforms and visualize the results. Results We present a Linux-based toolkit, RNA-combine, to automatically perform the quality assessment, downstream analysis of the transcriptome data generated from different sequencing platforms, including bulk RNA-seq (Illumina platform), single cell RNA-seq (10x Genomics) and Iso-Seq (PacBio) and visualization of the results. Besides, this toolkit is implemented with at least 10 analysis modules more than other toolkits examined in this study. Source codes of RNA-combine are available on GitHub: https://github.com/dongxuemin666/RNA-combine. Conclusion Our results suggest that RNA-combine is a reliable tool for transcriptome data processing and result interpretation for both bioinformaticians and non-bioinformaticians.


2022 ◽  
Vol 8 (1) ◽  
pp. 52
Author(s):  
Ricardo Franco-Duarte ◽  
Neža Čadež ◽  
Teresa Rito ◽  
João Drumonde-Neves ◽  
Yazmid Reyes Dominguez ◽  
...  

Clavispora santaluciae was recently described as a novel non-Saccharomyces yeast species, isolated from grapes of Azores vineyards, a Portuguese archipelago with particular environmental conditions, and from Italian grapes infected with Drosophila suzukii. In the present work, the genome of five Clavispora santaluciae strains was sequenced, assembled, and annotated for the first time, using robust pipelines, and a combination of both long- and short-read sequencing platforms. Genome comparisons revealed specific differences between strains of Clavispora santaluciae reflecting their isolation in two separate ecological niches—Azorean and Italian vineyards—as well as mechanisms of adaptation to the intricate and arduous environmental features of the geographical location from which they were isolated. In particular, relevant differences were detected in the number of coding genes (shared and unique) and transposable elements, the amount and diversity of non-coding RNAs, and the enzymatic potential of each strain through the analysis of their CAZyome. A comparative study was also conducted between the Clavispora santaluciae genome and those of the remaining species of the Metschnikowiaceae family. Our phylogenetic and genomic analysis, comprising 126 yeast strains (alignment of 2362 common proteins) allowed the establishment of a robust phylogram of Metschnikowiaceae and detailed incongruencies to be clarified in the future.


2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Bohu Pan ◽  
Luyao Ren ◽  
Vitor Onuchic ◽  
Meijian Guan ◽  
Rebecca Kusko ◽  
...  

Abstract Background Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. Results To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. Conclusions Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.


Lab on a Chip ◽  
2022 ◽  
Author(s):  
Lin Sun ◽  
Thomas Lehnert ◽  
Songjing Li ◽  
Martin A. M. Gijs

We present a new bubble-enhanced microfluidic approach for highly efficient DNA fragmentation, suitable for next generation sequencing platforms. Improved on-chip performance arises from acoustic streaming generated by oscillating bubble interfaces.


2021 ◽  
Author(s):  
Twinkle Soni ◽  
Madhvi Joshi ◽  
Ramesh Pandit ◽  
Chaitanya Joshi ◽  
Damer P. Blake

The use of antibiotics in human medicine and livestock production has contributed to the widespread occurrence of antimicrobial resistance (AMR). Recognizing the relevance of AMR to human and livestock health, it is important to assess the occurrence of genetic determinants of resistance in medical, veterinary, and public health settings in order to understand risks of transmission and treatment failure. Advances in Next Generation Sequencing (NGS) technologies have had a significant impact on research in microbial genetics and microbiome analyses. Now, strategies for high throughput sequencing from panels of PCR amplicons representing known AMR genes offer opportunities for targeted characterization of complex microbial populations. Aim of the present study was to compare the Illumina MiSeq and Ion Torrent S5 Plus sequencing platforms for use with the Ion AmpliSeqTM AMR Research Panel in a veterinary/public health setting. All samples were processed in parallel for the two sequencing technologies, subsequently following a common bioinformatics workflow to define the occurrence and abundance of AMR gene sequences. Regardless of sequencing platform, the results were closely comparable with minor differences. The Comprehensive Antibiotic Resistance Database (CARD), QIAGEN Microbial Insight - Antimicrobial Resistance (QMI-AR), Antimicrobial resistance database (AR), and CARD-CLC databases were compared for analysis, with the most genes identified using CARD. Drawing on these results we describe an end-to-end workflow for AMR gene analysis using NGS.


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