To test for BRCA gene mutations, a patient sample, typically a blood sample, is sent to a lab that specializes in genetic testing. The goal of genetic testing is to examine the DNA sequence of the gene to determine if any harmful mutations are present. Traditionally, this has involved amplifying many copies of the gene of interest ( BRCA1 or BRCA2 in this case) using the polymerase chain reaction (PCR), followed by Sanger sequencing using fluorescently-labeled nucleotides to determine the DNA sequence. While Sanger sequencing is still considered the gold standard to confirm DNA mutations, it can be time-consuming to sequence large genes. The length of a typical Sanger sequencing read is approximately 1 kb, while the BRCA1 gene is 5.6 kb in length and BRCA2 is 10.3 kb (Nicolussi et al., 2019).
Recent advances in DNA sequencing technology have allowed scientists to utilize next generation sequencing (NGS) as an alternative approach to traditional Sanger sequencing methods for detecting genetic mutations (Nicolussi et al., 2019, Figure 2). While Sanger sequencing reads a single DNA sequence at a time, an NGS approach first breaks the DNA into many smaller fragments (200-500 bp), to which specific identifier tags are added at the ends to create a library of DNA fragments from the gene of interest. Each DNA library uses unique tags that tie the sequences back to specific genes from a specific patient. Millions of DNA fragments from multiple sample libraries (e.g. multiple patients being tested for both BRCA1 and BRCA2 mutations) can then be amplified and sequenced at a time using a high-throughput NGS platform.
The DNA sequences of overlapping fragments are then aligned using software to assemble the DNA sequence of the entire gene. Because NGS can sequence many DNA samples simultaneously, using NGS technology for genetic testing can increase the number of different genes and patient samples that can be tested while decreasing turnaround time and sequencing cost per sample, enabling doctors and patients to receive BRCA genetic testing results more quickly and cost effectively.