SRC : Short Read Sequencing

What's the difference?

LRS allows for the retrieval of much longer (>10,000bp) sequencing reads than widely-used SRS systems (75-300bp). Some long-read sequencing (LRS) platforms have produced sequence reads of 882,000bp 1,with some user groups reporting reads of over 2,000,000bp (2MB) 2; however, read lengths of 10,000-100,000bp are more common.

Currently, the two dominant producers of ‘true’ long-read sequencing technologies are Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (Nanopore). Both have developed platforms for ‘real-time’ sequencing of nucleic acids (DNA and RNA) that is faster than current short-read technologies.

Benefits of LRS

• Genome assembly: The human genome is over 3 billion DNA base pairs in length and contains many repetitive stretches of genetic code. Like a complex jigsaw, reassembling the genome from short reads can be challenging, as many fragments look highly similar without additional context. Long-read data can make this task simpler as the reads are more likely to look distinct, allowing them to be assembled together with less ambiguity and error. Improvements in genome assembly are helping to close gaps in our knowledge of the genome and allow for a better understanding of the genetic causes of disease.
• Variant detection: Some features of individual genomes are particularly difficult to detect and quantify with SRS technologies, for example: large and complex rearrangements, large insertions or deletions of DNA, repetitive regions, highly polymorphic regions, or regions with low DNA nucleotide diversity. Long reads can span across larger parts of these regions, so are able to detect more of these variants, which may be clinically relevant. LRS may also enhance the ‘genome-wide’ detection of certain variants 3.
• Haplotype phasing: In areas such as reproductive medicine it can be useful to know whether genetic variants exist on the same copy of the chromosome. This can be determined using a process known as haplotype phasing. Long reads are able to provide the long-range information for resolving haplotypes without additional statistical inference, maternal/paternal sequencing, or sample preparation, as is required for an approximation of phasing using SRS.
• Portability: In contrast to other sequencing platforms, Nanopore’s devices rely on detecting electronic rather than optical signals. This allows them to design devices as small as a memory (USB) stick, making them highly portable. Many other sequencers, including the vast majority of SRS systems, are large desktop or free-standing machines. Nanopore’s MinION device has been used to sequence samples in the field during the Ebola and Zika virus outbreaks and has even been used in space.
• Real-time sequencing and speed: Compared to the fixed run times of SRS systems, both PacBio and Oxford Nanopore offer faster sequencing runs. PacBio provides options for rapid sequencing that can be completed in <24hours, from sample preparation to analysis. Nanopore technologies permit real-time analyses and allow experimental run time to be determined by the user, giving the user the ability to track data collection and begin analyses as desired. This provides additional flexibility and speed, and removes the need for batch sequencing of multiple samples which is currently required for cost-effective SRS. It is particularly useful when examining small genomes (such as those of many pathogens) or specific genomic regions
• Other ‘omics: Long-read technologies have been used to directly sequence RNA. They may also allow simultaneous detection of epigenetic modifications (chemical modifications to DNA/RNA that affect how genes are expressed), although additional bioinformatic interpretation is required. Separate sequencing runs need to be performed to retrieve this information using current SRS systems

References PHG Foundation