Whole Genome Sequencing

How do you sequence an entire genome?

The first genome to be fully sequenced was the genome of the bacterium that causes bacterial influenza, Haemophilus influenzae. This bacterial genome was sequenced in 1995, using shotgun sequencing, which breaks the genome into small DNA pieces that are cloned into bacteria for growth, isolation, and sequencing. Sequences are then reassembled into the full genome using bioinformatics tools [1]. When the Human Genome Project attempted to sequence the first human genome in 2001, shotgun sequencing was also used, but, since the human genome is so much larger than a bacterial genome, the entire human genome could not be fully sequenced. Although faster than other sequencing methods at the time, it still took over ten years to fully sequence the human genome using shotgun sequencing.

Next generation sequencing (NGS) is a much faster sequencing approach that does not require cloning DNA fragments. Instead, DNA is extracted from an organism’s tissue and fragmented, then sequencing libraries are created by adding adapters that are later recognized by the sequencing platform. The libraries are then loaded onto the sequencer which uses a platform-specific technology to detect nucleotides one by one. Using NGS, a human genome can be sequenced in just a single day [2].

What is the difference between WGS and NGS?

WGS is an approach to sequencing the entire DNA sequence of an organism, while NGS is one of several sequencing technologies (as described below):

• Sanger sequencing: also known as chain termination or Dideoxy sequencing, Sanger sequencing requires four different PCR reactions (one per A, T, C, G) that are then heat denatured and separated by gel electrophoresis; bands on the gel are visualized and ordered to determine the DNA sequence
• Shotgun sequencing: randomly shears DNA sequences into smaller fragments and computationally assembles them by looking for overlaps to piece the whole sequence back together
• NGS: also known as massively parallel sequencing, NGS is a second-generation, high-throughput sequencing technology; single DNA strands are adhered to a solid surface and as the second strand is built, a fluorescent signal (e.g., Illumina platforms) or a pH measurement (Ion Torrent platforms) indicates the sequence of DNA bases
• Nanopore sequencing: a third-generation sequencing technology that uses protein nanopores to determine DNA sequences; when DNA passes through the nanopore, a current change occurs, with each base associated with a slightly different change

Whole Genome Sequencing workflow

Whole genome sequencing can be performed on as little as 1 ng of DNA that has been extracted from a target tissue or cell samples. Libraries are then prepared using one of several available library preparation kits and adapters which fragment the DNA then add adapters to the ends of the resulting pieces. Most sequencing libraries will contain indexing barcodes―short, fixed DNA sequences―unique to each sample, enabling multiple samples to be sequenced at one time (i.e., multiplexing). These barcodes also allow sequencing libraries to be separated during the sequence analysis step. Currently, there are companies and research cores that offer a wide range of sequencing services. A variety of free bioinformatics tools are available to analyze your data as well.