Microbial Whole Genome Sequencing is a critical approach for sequencing the entire microbial genomes, as well as for comparing multiple reference genomes to mapped genomes of new organisms. Sequencing entire bacterial, viral, or other microbial genomes is important for the generation of accurate reference genomes, microbial identification, and other comparative genomic studies.
Compared with conventional approaches like PCR, Whole Genome Sequencing does not require labor-intensive cloning and mapping steps. Hence, it is time- and cost-effective. Moreover, this high-throughput sequencing approach allows the sequencing of numerous samples at the same time through the courtesy of multiplexing.
Specifications: DNA Sample Requirements
|Library Type||Sample Type||Amount (Qubit)||Volume||Concentration||Purity (NanoDropTM/
|Microbial whole genome library (350bp)||Genomic DNA||≥200 ng||≥20 μL||≥10 ng/μL||A260/280=1.8-2.0;
|Microbial whole genome library
|Genomic DNA||≥1.2 μg||≥20 μL||≥10 ng/μL|
Specifications: Sequencing Parameters and Analysis Contents
|Platform Type||Illumina NovaSeq 6000|
|Read Length||Paired-end 150 bp|
|Recommended Sequencing Depth||≥ 100x for bacterial genomes|
|≥ 50x for fungal genomes|
|Standard Data Analysis|
The first step of the project workflow involves the sample quality control (Sample QC) to ensure that your samples meet the criteria of the Microbial WGS technique. Then, the appropriate library is prepared according to your target organism and subsequently tested for its quality (Library QC). Next, a paired-end 150 bp sequencing strategy is used to sequence the samples and the resulting data go through quality data control (Data QC) to guarantee the quality of the resulting data. Finally, bioinformatics analyses are performed and publication-ready results are provided. The following flowsheet describes the step-by-step protocol our Microbial WGS technique follows.
Preparation of sample is followed by the DNA library preparation which is verified for quality and yield. Genomic DNA is fragmented with a size of 350 bp which is narrowly size selected by sample purification beads. The selected fragments are then end polished, A-tailed, and ligated with the full-length adapter. Illumina PE150 technology is employed to sequence the sample and the final stage involves the bioinformatics analysis.
Featured Publications of Microbial Whole Genome Sequencing
mSystems Date: 12 January 2021IF: 6.663DOI: https://doi.org/10.1128/mSystems.01161-20
Science of the Total Environment Date: 20 june 2021IF:6.551DOI: https://10.1016/j.scitotenv.2021.145767
food chemistry Date: 15 November 2020IF: 6.306DOI: https://10.1016/j.foodchem.2020.127316
Genomics Date: 23 February 2021IF: 6.205DOI: https://doi.org/10.1016/j.ygeno.2020.04.018
Chemosphere Date: 25 October 2020IF: 5.108DOI: https://doi.org/10.1016/j.chemosphere.2020.127291
Journal of Global Antimicrobial Resistance Date: 23 December 2020IF: 4.035DOI: https://10.1016/j.jgar.2020.08.002
Nature Communications Date: 25 june 2020IF:14.919DOI: https://10.1038/s41467-020-16990-3
SNP Mutation Frequency
Single nucleotide polymorphism (SNP) refers to a variation in a single nucleotide that can occur at some specific position in the genome, including transition and transversion of a single nucleotide.
Taken the T: A>C: G mutations as an example, this category includes mutations from T to C and A to G. When T>C mutation appears on either of the double-strand, the A>G mutation will be found in the same position of the other chain. Therefore the T>C and A>G mutations are classified into one category. Accordingly, the whole genome SNP mutations could be classified into six categories. The frequency of each type is shown in Figure X.
The x-axis represents the number of SNPs and the y-axis indicates the mutation types.
Length Distribution of CDS-located InDels
InDel refers to the insertion or deletion of ≤ 50 bp sequences in the DNA. The results demonstrate several peaks present at certain InDel lengths. Non-frameshift InDels exert a smaller effect on the genome as compared to frameshift InDels.
The x-axis represents the proportion of the InDels with a certain length, and the y-axis indicates the length of the InDels.
Length Distribution of SVs
Structural variants (SVs) are genomic variations with mutations of relatively larger size (>50 bp), including deletions, duplications, insertions, inversions, and balanced translocations.
The x-axis represents the proportion of the SVs with a certain length range, and the y-axis indicates the certain length range of the SVs. Note, the length of DNA insert in library construction impacts the SVs detection greatly.
The distribution of CNVs on the genome
Copy-number variation (CNV) is a type of structural variation that happens when a DNA fragment is present in variable copy number in comparison to a reference genome. It pinpoints the deletions and duplications in the genome.
The x-axis represents samples and the y-axis indicates the number of CNVs in a different region.
Visualization of Variation
For proper visualization of the structural variations in the whole genome, we present mutation types with Circos:
(1) for SNP/InDel type, the density distribution is drawn;
(2) for SV/CNV type, the location and size are drawn.
From outer to inner: chromosome, SNP, InDel, CNV duplication, CNV deletion, SV insertion, SV deletion, SV invertion, SV ITX, SV CTX.
*Please contact us to get the full demo report.