Introduction
Third-generation sequencing (also knowns as long-read sequencing) is a technology that reads the nucleotide sequences at single-molecule level. Comparing to existing methods (such as Illumina platform, BGI complete genomics platform, Thermofisher Ion Torrent platform, Qiagen GeneReader Platform) that infer nucleotide sequences by amplification and synthesis (SBS: sequencing by synthesis), the third-generation sequencing gives three major advantages:
- - Rapid sequencing pipeline. Sequencing does not base on the synthesis, no clonal amplification step (which is for amplifying signal) in certain sequencers.
- - Improving de novo genome assembly, transcriptome gene isoform identification, fusion gene analysis, big-size genome structure variation analysis. All these benefits from the long reads.
- - Direct sequencing can identify base modifications in DNA and RNA.
Currently, we are offering Oxford Nanopore sequencing using two major systems: GridION (150 Gb data) and PromethION (4800/9600 Gb data), which enable low to high-throughput long-read sequencing of both DNA and RNA samples. For high molecular weight DNA (HMW-DNA) samples, read lengths of several hundred kb to a maximum of 2.3 Mb can be reached with ultra-long-read protocols. The Nanopore sequencing data greatly enable de novo genome assemblies and structural genomic variant and transcriptome studies. To learn more about the technology, please see the HOW-IT-WORK page from Oxford Nanopore.
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Quick Biology ‘s Nanopore sequencing offers advantages in all areas of research. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analyzing proteins.
Sample Requirements
- - Human, animals, plants, and microorganisms
- - Tissue, Whole Blood, and Cell lines, we will perform DNA extraction from sources.
- - Genomic DNA samples (DNA should be > 5 ug, at a concentration of 100ng/µl in 50µl volume in 10 mM TRIS (pH=8.0-8.4); OD260/280 and OD260/230=1.8~2.0; free of enzymatic inhibitors). For long-read sequencing genomic DNA samples isolated with spin column protocols ( e.g. Qiagen DNeasy) are sufficient. For super-long-read sequencing, we require high molecular weight DNA samples with fragment sizes over 50KB.
- - RNA samples (For full-length cDNA sequencing we require >1 ug of total RNA in up to 20 ul of molecular biology grade water. The RNA samples need to be DNA-free and need to be accompanied by Bioanalyzer traces. The samples should have RNA-integrity scores (RIN-scores) of 8 or higher; OD260/280 and OD260/280=1.8~2.0; free of enzymatic inhibitors)
Bioinformatics analysis support
- - Genome assembly - Generate more contiguous genome assemblies using long sequencing reads (with additional HiC-seq reads suitable for chromosome scaffolding) for any sized genome assembly project, from small individual microbial genomes to high-throughput, population-scale sequencing of large genomes.
- - Structural variation - Calling structural variations on whole genome sequencing data obtained from Oxford Nanopore sequencing platforms.
- - Single nucleotide variants (SNVs) and phasing - Calling single nucleotide variants and indels, Phase SNVs and resolve compound heterozygosity
- - Chromatin conformation - Chromatin conformation capture (3C) techniques reveal genomic interactions in three dimensions. This can provide key information on the effect of chromatin structure on transcriptional regulation; the data can also be utilized to orient contigs, producing highly contiguous scaffolded assemblies.
- - Epigenetics - Directly identified DNA base modifications at nucleotide resolution, including 5mC, 5hmC, m6A, and BrdU, with detection of other natural or synthetic epigenetic modifications; Phase epigenetic DNA modifications or unambiguously assign RNA modifications to transcript isoforms using long sequencing reads.
- - Gene expression - Accurately characterize and quantify full-length transcripts at the isoform level using long sequencing reads
- - Splice variation - Accurately resolve and quantify full-length splice variants with long sequencing reads; Identify epigenetic modifications alongside nucleotide sequence through direct RNA sequencing
- - Fusion transcripts - Fusion transcripts can be sequenced end-to-end in single reads, enabling comprehensive characterization of fusions and their precise splice junctions.