Shotgun Metagenome Sequencing

In metagenomics, genomes from environmental samples are analyzed without the prior isolation and cultivation of individual species, and, therefore, it is a powerful technique for studying microbial communities in their natural habitat, with a broad range of applications.

 

The method enables microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments. Shotgun metagenomics also provides a means to study unculturable microorganisms that are otherwise difficult or impossible to analyze.

 

Unlike capillary sequencing or PCR-based approaches, next-generation sequencing (NGS) allows researchers to sequence thousands of organisms in parallel. With the ability to combine many samples in a single sequencing run and obtain high sequence coverage per sample, NGS-based metagenomic sequencing can detect very low abundance members of the microbial community that may be missed or are too expensive to identify using other methods.

 

At Nucleome our customers can rely on our expertise in NGS to help them explore the rich genetic repertoire of microbial communities, while also benefiting from our bioinformatics expertise to help identify the species, genes, and pathways represented in their samples. Nucleome provides metagenomic sequencing service with Illumina HiSeq platform and assembly-first strategy, and our bioinformatics analyses provide gene predictions, function annotations, and taxonomic annotations.

16S Ribosomal RNA (rRNA) Sequencing

The 16S ribosomal RNA (rRNA) sequencing is a common amplicon sequencing method used to identify and compare bacteria present within a given sample. The 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.

 

16s Amplicon sequencing is frequently used to identify and differentiate microbial species. Short (< 500 bp) hypervariable regions of conserved genes or intergenic regions are amplified by PCR, analyzed using NGS technology, and the resulting sequences are compared against microbial databases. We can provide this service on Illumina MiSeq/HiSeq 2500 PE 250bp read length as well as PacBio RS II.

 

For bacteria and archaea, the 16S rRNA gene is the most common target for amplicon sequencing. For fungi, three targets are generally used: the 18S rRNA gene, and two internal transcribed spacers (ITS) located between rRNA genes. These regions are usually sufficiently divergent to separate even highly related species, and can sometimes differentiate subspecies.

 

Our standard bioinformatics analyses include alpha-diversity analysis, OTU analysis, species annotation, beta-diversity analysis, and multi-variate statistical analysis. Applications range from identifying a single species in pure culture to characterizing the microbiota of animals or plants to comparing species diversity and population structure in various environmental sources or geographic regions. Our specialists can advise you on the appropriate analyses for your project.

Metatranscriptome Sequencing

Microbial transcriptome and metatranscriptome information is important for predicting resistance to specific antibiotics, understanding host-pathogen immune interactions, quantifying gene expression changes, and tracking disease progression. Next-generation RNA-Seq (RNA sequencing) of bacteria, viruses, and other microbes has become a standard method for analyzing transcriptome and metatranscriptome information.

 

Cellular RNA is extracted and converted to cDNA, which is used to prepare sequencing libraries. Sequence reads are then mapped back to the reference genome, providing qualitative information on features such as exon junctions and splicing sites, as well as quantitative transcript data that can be compared across many experimental sets.
Unlike hybridization-based methods such as microarrays, RNA-Seq enables unbiased strand-specific identification of common and novel transcripts. A wide dynamic range enables confident identification of both high and low expressors in a single bacterial, viral, or other microbial RNA-Seq experiment. Multiple samples can be processed at once with a streamlined workflow suitable for automation.

Microbial Genome Sequencing

De Novo Microbial Genome Sequencing

 

De novo whole-genome sequencing involves assembling a genome without the use of a genomic reference and is often used to sequence novel microbial genomes. Illumina sequencers provide high coverage but shorter read lengths of unparalleled raw read accuracy. We also use PacBio RS II or Sequel for its higher read length and read depth for high-quality draft and complete microbial genome assemblies.

 

Microbial Whole-Genome Resequencing

Nucleome provides microbial whole-genome resequencing services to sequence the entire genome of a bacteria, virus, or other microbe, and comparing the sequence to that of a known reference for detecting low frequency mutations, finding key deletions and insertions, and discovering other genetic changes among microbial strains.

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