Miga and colleagues initially sought a source of human genome material that contained identical pairs of chromosomes from only one parent, to avoid the added complication of assembling both a maternal and paternal genome. This condition was satisfied by a human cell line derived from a rare occurrence called a hydatidiform mole.

“The hydatidiform mole forms when something goes wrong during conception,” explained Tamara Potapova, PhD, a research specialist II in the Gerton Lab. “The egg’s (maternal) genome is lost, and the paternal genome gets duplicated, resulting in a genome with mostly identical pairs of chromosomes.”

Because hydatidiform mole tissue can easily become aneuploid (having extra or missing chromosomes), which would pose a problem for researchers having to contend with variable copies of certain regions, the need to identify a stable cell line, with a normal number of chromosomes, was urgent.

“Tamara’s expertise in imaging chromosomes has been a huge asset for the project,” Gerton said. “She has a talent for culturing and evaluating cell lines that are very difficult to work with.”

During the early stages of the project, and for the main Science paper, Potapova helped identify the CHM13 cell line as the most stable of the hydatidiform mole cell lines.

“At that point, Tamara became a cytogeneticist for the whole project,” Gerton said. “When it comes to the ground truth of assembled sequence, does it match what we see in the chromosomes by microscopy? Tamara is the person we go to when we ask those questions. The beautiful picture of spectral karyotyping, identifying all the chromosomes, on the T2T website—that’s hers.”

Potapova studies nucleolar organizing regions, which organize the nucleolus, a specialized compartment within the cell’s nucleus. These genetic regions consist primarily of ribosomal DNA, which has unique behavior relative to most other coding regions of DNA.

Because ribosomal DNA genes are repetitive and are present in multiple, nearly identical copies, assembling these regions of the human genome was previously impossible. New sequencing technologies that produced long and highly accurate stretches or "reads" of DNA made it possible for Adam Phillippy’s group to find a path through this "dark matter."

“Adam’s group used this long-read sequencing method because a single ‘read’ could span multiple repeats and also the neighboring regions. Untangling these reads so they could assign them to a particular chromosome was a very complex process,” said Potapova.

“A brilliant computer scientist in my lab, Sergey Nurk, PhD, developed new methods to squeeze every last bit of information out the latest sequencing data,” said Phillippy. “With his tools, it was like putting on a new pair of glasses. All of sudden we could see every region of the genome with unprecedented clarity.”

“We wanted to know with high certainty how many ribosomal DNA repeats each chromosome had in order to fill the gaps with the correct number of copies. That question was very hard to approach even by modern sequencing methods. But we could estimate the number of ribosomal DNA repeats on each individual acrocentric chromosome by fluorescence microscopy,” said Potapova.

For the paper, “we made chromosome preparations and marked the ribosomal DNA with fluorescent labels. We knew the total copy number of the repeats from sequencing and a special PCR technology called droplet digital PCR, and we could measure the fluorescence intensity of all ribosomal DNA locations in a chromosome preparation. From that, we could calculate the fraction of the total fluorescent signal that was present on every acrocentric chromosome and convert that to the number of copies of the repeats.”

“Jay Unruh, PhD, who is director of scientific data at the Stowers Institute, was very helpful with analyzing this imaging data, which is not trivial,” said Potapova. “We benefited greatly from all the advice and feedback that Jay and the Microscopy Center team contributed to our work on an ongoing basis.”

This unprecedented resolution enables scientists to ask new questions about ribosomal DNA, and more generally, acrocentric chromosomes. Scientists can ask how these chromosomal regions are inherited from parent to child and how they organize chromosomes in three dimensions. Because ribosomal DNA is crucial for cellular function, this information opens new doors for understanding how cells develop into tissues, and how health and disease depend on ribosomal DNA.

• Completing the sequence - Introduction
• Part 1: Seeing is believing
• Part 2: Insights from evolutionary biology
• Part 3: A ‘big data, AI, and genomics’ approach to identifying variation