Sequence assembly, binning, taxonomy assignment, composition profiling, etc.
HMW DNA from DNA extraction with mechanical microbial lysis
Entrust your samples to PacBio Certified Provider
Zymo Research Analysis Sequencing team gives you the tools and capability to untangle the mysteries of your metagenome. Our technical specialists ensure the ease of your ongoing discovery.
The Metagenomic Assembly sequencing service report provides binning results, compositional bar plots, and alpha-diversity plots. Our team of expert bioinformaticians are here to help you get the most out of your data.
View Sample ReportFragment size distribution of fecal DNA extracted with DNA extraction protocols using mechanical lysis. DNA extracted using ZymoBIOMICS 96 DNA MagBead Kit and Miniprep Kit.
| HiFi Reads | ||||||
|---|---|---|---|---|---|---|
| Sample Information | Total Base | Unique Molecular Yield (Gb) | >Q20 Reads | Yield | Mean Length | Median QV |
| Vaginal Microbiome Metagenomic | 760.98 | 70.11 | 3384650 | 20.55 Gb | 6072 | Q41 |
| Sequencing Platform | PacBio Sequel Ile |
|---|---|
| Sequencing Kit | SMRT Cell 8M |
| Target Amplicon | The full-length 16S rRNA gene is amplified with 27f (AGRGTTYGATYMTGGCTCAG) and 1492r (RGYTACCTTGTTACGACTT) |
| Accepted Sample Types | Extracted genomic DNA, or raw samples |
| DNA Extraction | |
| Library Prep | We follow PacBio recommended protocol (https://www.pacb.com/wp-content/uploads/Procedure-checklist-Amplification-of-bacterial-full-length-16S-rRNA-gene-with-barcoded-primers.pdf) |
| Quality Control | A positive control for DNA extraction (ZymoBIOMICS Microbial Community Standard, D6300) and a positive control for library preparation (ZymoBIOMICS Microbial Community DNA Standard, D6305) are included in each project as a quality control. Results are reported in the final project report. Negative extraction controls and negative qPCR controls are also monitored with real time PCR and can be analyzed upon request. |
| Sequencing Analysis | Amplicon Sequence Variant (ASV) based analysis with DADA2 is used to provide higher accuracy and better resolution of data. |
| Reference Database | As publicly available databases (e.g. Greengenes, UNITE) contain a multitude of errors, inconsistent annotations, and poor sequence quality, we utilized an in-house curated reference database. |
| Data Visualization | Absolute abundance boxplot, taxa abundance barplot, taxa abundance heatmap, ASV abundance heatmap, alpha diversity boxplot and rarefaction curves, beta diversity 3D Emperor plot, Taxa2ASV decomposer |
| Statistical Analysis | LEfSe analysis uncovers taxa/traits that are differentially distributed among defined sample groups. |
Metagenomic sequencing and analysis represent a groundbreaking approach in microbiology research, enabling the study of intricate microbial communities without the difficult task of isolating and cultivating individual organisms. Metagenomic assembly encompasses two key facets: wet-lab procedures, involving DNA extraction, library preparation, and sequencing, and dry-lab tasks, which include read quality enhancement, DNA sequence assembly, contig/scaffold binning, taxonomy annotation, gene prediction, and gene annotation. While assembly-independent pipelines offer avenues for taxonomy analysis without DNA sequence assembly, the assembly step becomes indispensable when the research goal extends beyond taxonomy analysis to encompass genome reconstruction, comprehensive gene retrieval, and organism-specific pathway exploration.
The objective of DNA sequence assembly is clear-cut: to attain extensive contigs/scaffolds while minimizing misassemblies and gaps, allowing for the reconstruction of complete genomes. In the context of pure bacterial culture genome assembly, the predominant challenge lies in repetitive sequences, e.g. multiple copies of ribosomal RNA sequences within a genome. However, in metagenomics, this process becomes exponentially challenging due to the varying sequencing depth/coverage of microbial genomes, the presence of closely related genomes (including multiple strains of a species), and horizontal gene transfers across phylogenetically distant genomes. Traditional short-read sequencing methods, such as Illumina sequencing, typically yield short contigs and require extensive computational resources, thereby limiting the advantages of DNA sequence assembly.
PacBio HiFi reads, generated through circular consensus sequencing (CCS) mode on PacBio long-read systems, have a size of up to 15-20 kb with an accuracy exceeding 99% (Q20 or better). By harnessing the potential of HiFi reads in conjunction with sophisticated bioinformatics pipelines, it is now feasible to directly derive hundreds of complete or high-quality genomes, known as metagenome-assembled genomes (MAGs), from complex metagenomic samples, such as human fecal specimens.
At the forefront of metagenomic analysis, Zymo Research offers a Certified PacBio Provider status, ensuring the meticulous handling of your samples. Our PacBio metagenomic sequencing and assembly service encompass the entire workflow, ranging from DNA extraction to metagenomic assembly and comprehensive analyses. We gladly accept high-molecular-weight DNA extracts, or alternatively, we can perform DNA extraction when provided with raw samples. Our use of ZymoBIOMICS DNA extraction kits guarantees impartial microbial lysis through mechanical bead beating while preserving high molecular weight (8-15kb). Our bioinformatics pipelines encompass adapter and quality trimming, DNA sequence assembly, binning, MAG taxonomy annotation, and taxonomy composition profiling. In the DNA sequence assembly phase, we employ state-of-the-art algorithms optimized for metagenomic assembly with HiFi reads, as recommended by PacBio. With us, you can have peace of mind, knowing that we will manage every aspect of your project with the utmost care and expertise.
