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Short-read sequencing involves sequencing short fragments of DNA, typically between 50–600 bp, and offers high data quality and sequencing depth at a low cost. Illumina short-read sequencing by synthesis (SBS) produces highly accurate reads 50 to 600 DNA bases long. Short-read SBS is easily scalable and can be targeted to focused parts of genomes up to full genomes.

Long-read sequencing can produce reads tens of thousands of bases long. Long-read sequencing has some advantages with sequencing genomes without a complete reference, identifying large structural rearrangements, and sequencing through low-complexity regions of a genome.

The main advantages of Illumina short-read sequencing are the high accuracy of sequencing by synthesis (SBS) chemistry, flexibility of assay designs from small panels up to full genomes, and scalability with a range of instruments and solutions for everything from investigating individual genomes to large-scale population initiatives. While short-read sequencing is able to sequence the vast majority of the human genome, there is a very small percentage with repetitive motifs, motifs with homology to other genomics regions, or large structural elements that can be difficult to resolve. Illumina mapped read technology provides long-distance genomic information that can help scientists resolve these challenging-to-map regions.

Learn more about mapped read technology

It can be advantageous to combine long-read data with complementary short-read information. Many long-read sequencing technologies have laborious workflows as well as highly variable results.^1-4 Short reads (typically 50–600 bp) offer high data quality and sequencing depth at low cost. With advanced NGS data analysis, short-read sequencing can generate whole-genome variant calls with outstanding accuracy. In addition, a small fraction of genomic regions can benefit from long-read information to improve resolution of difficult-to-map genes.

Linked-read sequencing is a category of methods that attempt to get the long-distance information of long-read sequencing with the accuracy of short-read sequencing. These methods modify long DNA templates to introduce a chemical tag or sequence barcode that is used during the analysis to map longer sequences.

The main downside to linked-read sequencing is that the template modification and additional analysis steps increase complexity and cost, which limits their usability and scalability for many labs. Long reads may still be advantageous when mapping genomes without a reference, accurately mapping in repetitive regions, phasing of variants, and identifying large structural variants.

Synthetic long-read sequencing involves tagging long DNA templates with unique sequence barcodes or chemical tags, as well as fragmenting the DNA. The DNA fragments are then sequenced on a short-read sequencing instrument and assembled into synthetic long reads using specialized bioinformatics software. The software uses the barcodes to map the DNA fragments back to the original long DNA templates.

The main drawback to synthetic long-read sequencing is that the DNA template modifications and additional computational analysis steps increase complexity and cost, which limits usability and scalability for many labs.