Revolutionizing the field

Located on Yale’s West Campus, Yale Center for Genome Analysis (YCGA) is a busy lab that sequences, on average, about 5,000 DNA samples each month. To accomplish this, the YCGA team uses state-of-the-art equipment, high-performance computers and sophisticated bioinformatics/artificial intelligence analytical tools.

The YCGA provides next-generation sequencing of DNA and RNA samples for Yale and non-Yale investigators, clinicians, and non- or for-profit organizations. Physicians can sequence a patient’s DNA to provide diagnosis and suggest treatments. Researchers can also study an extinct animal species using DNA extracted from its bones, and can even be helpful in solving centuries old mysteries. For example, to identify the artist who created a painting, a researcher sent scrapings from the painting to YCGA. The artist’s DNA was extracted from the sample and the sequencing results helped to identify the artist and approximate age of the canvas.

The information gathered from an individual’s genetic makeup assists clinicians and researchers in a variety of way, including advancing our understanding of how biology works, improving patient care, and contributing to personalized medicine.

Sequencing

“The ultimate biomedical goal of genomics is personalized medical treatment,” said Shrikant Mane, the Center’s director. “Sequencing has revolutionized the field of genomics by allowing for a rapid and cost-effective analysis of the entire genome.”

Sequencing is key to understanding the function of genes, allowing scientists, researchers, and clinicians to read the information in an individual’s genome. Sequencing is used in medical research to identify disease associations that can help with diagnosis, treatment, or drug discovery. In ecological research for biodiversity studies, it can be used to determine organisms in soil, water, or sewage. Sequencing is also employed to track the spread of pathogens “or in the case of the COVID-19 surveillance project we were involved with, to detect new variants,” said Irina Tikhonova, biotechnology associate.

Josephine Evan, biotechnology associate, prepares DNA samples for sequencing. DNA comes from biological samples that include blood, saliva, tissue, or cultured cells.

“As [sequencing] evolves, it will play an increasing role in diagnosing, treating, and preventing diseases.”

The sequencing process begins with extracting DNA or RNA from a biological sample, which can be blood, saliva, tissue (frozen or fixed), and cultured cells. Environmental samples can include soil, lake, river, or sea water, bacterial fungus, or plant tissue. After the extraction and purification process, the sample is a colorless, odorless, water-like liquid that potentially contains a vast amount of information that sequencing can help unlock.

Each month the YCGA sequences, on average, about 5,000 DNA samples.

“Most of the DNA or RNA we receive are from researchers and clinicians and have already been extracted. We take those and prepare the library — a collection of short DNA pieces — by cutting the long stretches of DNA into smaller fragments and adding adapters to each end,” said Tikhonova. “This is done by using high-frequency sound waves or enzymes. The adapters include a barcode for identification and allow sequencing multiple samples simultaneously.”

She and her team must carefully prepare the watery-looking samples for sequencing through a series of enzymatic and chemical reactions done by technicians and robotic systems, with exact precision. Next steps include amplification (producing multiple–sometimes millions–copies of one DNA sample), purification, and quality control.

Targeted Sequencing

“We don’t need to sequence the whole genome, just a targeted region. An example of targeted sequencing is screening for known cancer genes in cells,” said Tikhonova. “About 98% of the DNA in a genome is non-functional or unknown. For instance, it does not make your hair grow or organs work. Only 2% is useful, called the exome, which holds the protein-coding genes and controls about 99% of human function; that is the portion we sequence.”

Matthew Brandt, biotechnology associate, doing the final prep before loading DNA samples into the sequencing machine.

Next, these carefully prepared samples are handed off to Chris Castaldi, research associate, and his team, who will do the final preparation before loading them into the Illumina NovaSeq X Plus (X Plus). These next steps include combining the samples into a single well and then applying them to the glass surface of a flow cell. The flow cells are inserted into the X Plus which will scan them repeatedly, gathering data over a two-day period. Once that cycle is complete the data is sent electronically via email to the requester.

Flow cells containing DNA samples are inserted into the NovaSeq X-plus.

“We pool all the samples, load them onto the sequencer, and hit start. The adapters added during the library prep process bind the DNA to the flow cell’s surface, allowing the sequencing stage to occur and enabling the NovaSeq X-plus to read the information contained in the samples.” said Castaldi.

Improving patient care

“Most of the requests we receive are from researchers or clinicians who have a question about a disease they are studying or a sick patient they want to treat,” said Castaldi. “Sequencing can help by hopefully answering their questions through the data we send them.”

During the sequencing process, the machine displays graphs and quality metrics that the YCGA team can watch in real time.

“The information gathered from an individual’s genetic makeup assists clinicians and researchers in a variety of ways: to advance our understanding of how biology works, improve patient care, and contribute to personalized medicine,” added Mane. “As it evolves, it will play an increasing role in diagnosing, treating, and preventing diseases.”