Dataset Information

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio.

ABSTRACT:

Background

A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300?bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform.

Results

A total of 122.6?Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6?Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform.

Conclusions

The oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. This trial is registered with NCT02341352.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7748900 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio.

Zhang Jie J Su Lingkai L Wang Yuan Y Deng Shuli S

The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale 20201211

<h4>Background</h4>A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. <i>Material and Methods</i>. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then ...[more]

PMID: 33381248

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Project description:BackgroundLarge insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries.ResultsA Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~?8 kb) to be fragmented into 2-3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5-10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations.ConclusionsWe developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

Project description:The incorporation of plant residues into soil can be considered a keystone sustainability factor in improving soil structure function. However, the effects of plant residue addition on the soil microbial communities involved in biochemical cycles and abiotic stress phenomena are poorly understood. In this study, experiments were conducted to evaluate the role of raw garlic stalk (RGS) amendment in avoiding monoculture-related production constraints by studying the changes in soil chemical properties and microbial community structures. RGS was applied in four different doses, namely the control (RGS0), 1% (RGS1), 3% (RGS2), and 5% (RGS3) per 100 g of soil. The RGS amendment significantly increased soil electrical conductivity (EC), N, P, K, and enzyme activity. The soil pH significantly decreased with RGS application. High-throughput Illumina MiSeq sequencing revealed significant alterations in bacterial community structures in response to RGS application. Among the 23 major taxa detected, Anaerolineaceae, Acidobacteria, and Cyanobacteria exhibited an increased abundance level. RGS2 increased some bacteria reported to be beneficial including Acidobacteria, Bacillus, and Planctomyces (by 42%, 64%, and 1% respectively). Furthermore, internal transcribed spacer (ITS) fungal regions revealed significant diversity among the different treatments, with taxa such as Chaetomium (56.2%), Acremonium (4.3%), Fusarium (4%), Aspergillus (3.4%), Sordariomycetes (3%), and Plectosphaerellaceae (2%) showing much abundance. Interestingly, Coprinellus (14%) was observed only in RGS-amended soil. RGS treatments effectively altered soil fungal community structures and reduced certain known pathogenic fungal genera, i.e., Fusarium and Acremonium. The results of the present study suggest that RGS amendment potentially affects the microbial community structures that probably affect the physiological and morphological attributes of eggplant under a plastic greenhouse vegetable cultivation system (PGVC) in monoculture.

Dataset Information

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio.

Background

Results

Conclusions

Publications

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio.

Similar Datasets

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