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ObjectiveTo evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests.MethodsPatients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms.ResultsThirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs. 93.5% and 42.9 vs. 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium, and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs. 78.6%, 36.4% vs. 90.9%, and 50% vs. 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs. 77.8% and 42.9% vs. 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/42) based on pathogen diagnosis.ConclusionBoth platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection.

There is currently international interest in applying DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of four candidate plant DNA barcoding regions [rbcL, matK, trnL-F, and internal transcribed spacer (ITS)] in seven genera of Gramineae including Agropyron, Bromus, Elymus, Elytrigia, Festuca, Leymus, and Lolium. Fourteen accessions were analyzed, and matK and ITS showed the highest species, subspecies, and variety discriminatory power, each resolving 11 accessions. Species discrimination using rbcL and trnL-F was lower, resolving 7 and 8 accessions, respectively. Subspecies and variety discrimination using rbcL and trnL-F could not identify 4 accessions of Agropyron. A technical system can be established using the proposed DNA barcode to rapidly and reliably identify the seven genera of Gramineae. This study serves as a “useful reference” for identifying the genetic diversity of grass germplasm resources. DNA barcoding can be utilized to uncover the relatives of different species within the same family or between different families. It can also be used to determine the related groups of important herbage, turfgrass, and crops and provide crucial background information for discovering excellent genes and improving existing crop varieties.

Sweet sorghum (Sorghum dochna) is a high-quality bio-energy crop that also serves as food for humans and animals. However, there is little information on the genomic characteristics of S. dochna. In this study, we presented a high-quality assembly of S. dochna with PacBio long reads, Illumina short reads, high-throughput chromosome capture technology (Hi-C) sequencing data, gene annotation, and a comparative genome analysis. The results showed that the genome of S. dochna was assembled to 777 Mb with a contig N50 of 553.47 kb and a scaffold N50 of 727.11 kb. In addition, the gene annotation predicted 37,971 genes and 39,937 transcripts in the genome of S. dochna. A Venn analysis revealed a set of 7,988 common gene annotations by integrating five databases. A Cafe software analysis showed that 191 gene families were significantly expanded, while 3,794 were significantly contracted in S. dochna. A GO enrichment analysis showed that the expanded gene families were primarily clustered in the metabolic process, DNA reconstruction, and DNA binding among others. The high-quality genome map constructed in this study provides a biological basis for the future analysis of the biological characteristics of S. dochna, which is crucial for its breeding.

Gene mutations linked to lignin biosynthesis are responsible for the brown midrib (bm) phenotypes. The bm mutants have a brown-reddish midrib associated with changes in lignin content and composition. Maize bm1 is caused by a mutation of the cinnamyl alcohol dehydrogenase gene ZmCAD2. Here, we generated two new bm1 mutant alleles (bm1-E1 and bm1-E2) through EMS mutagenesis, which contained a single nucleotide mutation (Zmcad2-1 and Zmcad2-2). The corresponding proteins, ZmCAD2-1 and ZmCAD2-2 were modified with Cys103Ser and Gly185Asp, which resulted in no enzymatic activity in vitro. Sequence alignment showed that CAD proteins have high similarity across plants and that Cys103 and Gly185 are conserved in higher plants. The lack of enzymatic activity when Cys103 was replaced for other amino acids indicates that Cys103 is required for its enzyme activity. Enzymatic activity of proteins encoded by CAD genes in bm1-E plants is 23–98% lower than in the wild type, which leads to lower lignin content and different lignin composition. The bm1-E mutants have higher saccharification efficiency in maize and could therefore provide new and promising breeding resources in the future.

Objective: To evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests. Methods: Patients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms. Results: Thirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs. 93.5% and 42.9 vs. 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium, and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs. 78.6%, 36.4% vs. 90.9%, and 50% vs. 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs. 77.8% and 42.9% vs. 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/ 42) based on pathogen diagnosis. Conclusion: Both platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection. [ABSTRACT FROM AUTHOR]

Sheepgrass (Leymus chinensis) is a high-quality cool-season forage crop used as pasture and hay for livestock feeds. The presence of lignin in cell walls, however, impairs forage digestibility of such lignocellulosic feedstock. Here, the structural characterization and cell wall composition of sheepgrass internodes were studied, and a progressive increase in cell wall lignification was observed with internode maturation. Lignin composition analysis further revealed a gradual accumulation of guaiacyl and syringyl lignin units during internode development. Consistently, the transcript abundance of lignin-related genes was upregulated in mature internodes, suggesting their potential roles in lignin biosynthesis. Furthermore, the effects of cell wall composition and lignification extent on biomass saccharification efficiency were examined in sheepgrass. The results showed that lignin content, guaiacyl and syringyl lignin unit levels inversely correlated with cell wall digestibility, indicating that lignin is a crucial obstacle for utilizing sheepgrass feedstock. The baseline information obtained in this work will facilitate establishment, grazing management, harvesting and feedstock utilization of sheepgrass in future. [ABSTRACT FROM AUTHOR]