Researchers continue to face challenges in finishing the genomes, characterizing variants, and recognizing the role of key biological markers given the dramatic progress of NGS technology. In many areas of research, accurate read lengths of 1,000 base pairs (bp) or longer in a single sequencing reaction are important for investigators.
Genephenomics implements PacBio RS II / Oxford Nanopore Technology to overcome the DNA strand's long reads (up to 10 kb), enabling structural and cell type variability not explained by other sequencing technologies to be observed. The versatility of long read sequencing systems in Genephenomics allows you to easily switch between platforms and applications as the changing research environment changes research needs.
Sequencing Platforms
- PacBio RS II / PacBio Revio
- MinION, PromethION
Applications
Genome Finishing & De novo Assembly
- Support of multiple applications: genome ending, metagenomics, de novo assembly, meta-assembly with long & short reads
- High-quality reads of up to 30kb
Pacbio Sequencing
- Explores the distinct halotype quality of two homologous chromosomes
- One assay to process over 94 per cent of heterozygous SNPs and InDEls
- Easy push-button analysis within one day
Sequencing Platforms
Single Molecular Real-Time (SMRT) sequencing employs a specialized flow cell with several thousand individual picolitre wells with transparent bottoms— zero-mode waveguides (ZMW). The polymerase is fixed to the well's bottom and enables the DNA strand to advance through the ZMW. As a result, a single molecular could be the focus of the system. SMRT sequencing allows real-time visualization of fluorescently tagged nucleotides that are synthesized along individual molecules of the DNA template. When the template and polymerase dissociate, the sequencing reaction terminates. The PacBio instrument's average read length is about 2 kb, and some reads may be more than 20 kb. Longer readings are particularly useful for assembling new genomes de novo.
Highly repetitive elements present in eukaryotic and prokaryotic genomes pose a challenge to the assembly of genomes and make it difficult to study repetitive sequences in detail. Long-read sequencing offers reads spanning hundreds or thousands of kilobases (kbs), which can cover complex or repetitive regions with a single continuous reading, allowing these broad structural features to be solved. Besides significantly longer and highly accurate DNA sequences from individual unamplified molecules, it can also exhibit where methylated bases exist, thereby supplying functional information about the genome-encoded DNA methyltransferases. PacBio SMRT sequencing has unique advantages in the genomics, metagenomics, transcriptomics and epigenetics of de novo studies.
Nanopore sequencing
The nanopore based DNA and RNA sequencing technology was developed by Oxford Nanopore Technologies. The Nanopore sequencer is known to be compatible with a range of input materials, including genomic DNA, DNA amplified, cDNA, and RNA. Nanopore biomolecular sequencing technology has wide applications in the life sciences, Identification of pathogens, monitoring of food safety, genomic analysis, monitoring of the metagenomic environment and characterisation of bacterial antibiotic resistance.
The Nanopore sequencer has some highlighted advantages when opposed to the conventional workflow.
- Ultra-long reading lengths of several hundred kb may be sequenced in a single continuous reading for high molecular weight samples of DNA (HMW-DNA) The data on nanopore sequencing boost de novo genome assemblies and structural genomic variant and transcriptome studies considerably.
- The nanopore is holes of a nano-scale creating gateways through membranes in nature. A nanopore moves through nanopores an ionic current, and tests the current changes. We cause disruption in the current when molecules such as DNA or RNA pass through the nanopores. You can use the knowledge about the current change to classify the molecule. The native strand of interest is sequenced directly, without optics or amplification. Different kinds of library preparation protocols allow the direct sequencing of DNA / RNA with epigenetic information.
Advantages of PacBio SMRT or ONT Sequencing
- Long-Read metagenomic sequencing
- Bacterial whole genome de novo sequencing
- Fungal whole genome de novo sequencing
- Human genome PacBio SMRT or ONT sequencing
- Full-length 16S/18S/ITS rRNA sequencing
- The bioinformatics pipeline involves reference or de novo assembly, identification of specific modifications, generation of consensus between single molecules, amplicon analysis, sequence alignment with variant detection. And there's more data mining available according to your specific needs.
