Modern genomics seems to be undergoing a shift from using short-read technologies to sequence large numbers of genomes with the aim of detecting SNPs, to sequencing fewer genomes using long-read technologies that can resolve more complex events, e.g., structural rearrangements, copy number variations, and repeat expansions.
Long-read sequencing technologies provide highly accurate sequencing data and they are gaining traction within the following application areas:
- Long-read sequencing can be used to generate synthetic long reads to support de novo assembly and genome finishing applications.
- Genome sequencing. Long-read technologies make it possible to sequence previously challenging genomes, e.g., those with many stretches of highly repetitive elements.
- Genome phasing. This refers to the job of assigning alleles to the paternal and maternal chromosomes from which they were inherited. Long-read sequencing permits whole genome phasing studies to identify haplotypes (co-inherited alleles), and to phase de novo
- RNA studies. Long-read technologies make it possible to sequence whole transcripts and perform direct RNA sequencing (dRNA-seq). In dRNA-seq, RNA molecules are sequencing directly without the need for modification, e.g., enrichment, conversion to cDNA or PCR amplification. As such, dRNA-seq bypasses biases associated with RNA processing steps, while preserving important epigenetic and splice site information.
Good Quality DNA Is Everything!
One of the challenges we hear about when we speak with customers working with long-read DNA sequencing is DNA quality. High-quality, intact high molecular weight (HMW) DNA is a prerequisite for all long-read technologies. What is considered to be HMW DNA differs somewhat depending on field but in general the range is between 50 kb and > 300 kb.
Nordic BioSite offers a dedicated HMW DNA isolation kit from Zymo Research. The Quick-DNA HMW MagBead Kit is a 96-well format that yields ultra pure and highly concentrated HMW DNA up to 150 kb in length from any sample type, including biological fluids, cells, solid tissue, and environmental samples. Isolated DNA is ready for long-read sequencing on third-generation sequencing platforms such as Nanopore and PacBio SMRT Sequencing without any further processing.
Contact us to get your hands on a sample and try the kit for yourself.
Quality Control
Given the obvious importance of HMW weight DNA to long-read sequencing, any quality control protocol must provide information on DNA intactness as well as concentration and purity. We recommend using capillary electrophoresis e.g., a BioAnalyzer, to obtain accurate data about the aforementioned quality parameters.
Alternatively, it is possible to use a combination of approaches to get a clear picture of DNA quality, e.g., spectrometry to assess concentration and purity and agarose gel electrophoresis to assess DNA intactness, albeit qualitatively i.e. smearing indicates degradation but poor resolution may make it difficult to rank samples according to their intactness.
Core facilities and service providers that perform long-read sequencing usually have quality and concentration thresholds for input DNA so make sure to check with these ahead of time so you know what you are aiming for with your samples.
Handle With Care!
Some sample types are particularly easy when it comes to isolating high-quality HMW DNA, e.g., blood. However, with good handling it should be possible to get high-quality starting material from most sample types. Remember that what you do with your sample before you begin to use any isolation kit is just as (if not more) important as the kit itself.
In general, it is best to start with fresh material for long-read sequencing. If it is not possible to process your samples immediately after collection, it is vital to store them correctly to prevent nucleic acid degradation by nucleases present in the samples. Flash freezing in liquid nitrogen followed by storage at -80 °C or preservation in suitable stabilisation buffers, e.g., DNA/RNA Shield from Zymo Research is highly recommended to minimise DNA degradation in samples until DNA isolation and long-read sequencing is carried out.
Avoid multiple freeze-thaw cycles and any rough handling that may create turbulence and lead to unwanted DNA damage.
Beyond sample storage and preservation, good general handling and care in the working area can go a long way when working with HMW DNA or indeed any nucleic acid specimen. We have covered these in more detail in one of our earlier blog posts about general tips for NGS success.
Get in Touch!
At Nordic BioSite, we believe in finding the right solution based on your exact experimental goals and setup. Whether you are starting out in long-read sequencing or you are looking for ways to improve your workflow, we welcome you to get in touch with us by writing an email to info@nordicbiosite.com.