Promoting international collaboration through highly
accurate genome analysis and the genome bank project

NAKAMURA, Yasukazu Professor

Genome Informatics Laboratory
Department of Informatics

Professor Nakamura graduated from the Faculty of Agriculture, Shinshu University and completed his master’s course at the Graduate School of Agriculture, Kyoto
University. After serving as an engineer at the Yamanashi Agricultural Research Center, he entered the doctoral course in Genetics at the School of Life Science,
The Graduate University for Advanced Studies (NIG). After graduating, he assumed the position of research scientist at the Kazusa DNA Research Institute in
1996. He was promoted to be head of the laboratory at the same institution in 2006. He became a Professor at the National Institute of Genetics in January 2009 in
the Genome Informatics Laboratory at the then Bioinformation Research Center. Currently, he is a Professor and Project Research Scientist at the Genome
Informatics Laboratory at NIG. He also serves as Head of the International Affairs Division, Bioinformation and DDBJ Center.

"The Genetic code consists of four nucleotides, which are common to both liverworts and humans." — Having
carefully analyzed the entire genome of diverse organisms, Professor Yasukazu Nakamura has been fascinated bygenome analysis, which can be performed with any biological species. In the course of conducting research at various
levels within industry, government, and academia, Professor Nakamura is now in charge of an international
collaboration to expand the nucleotide sequence data bank project as the representative of the National Institute of
Genetics.

The twists and turns ofbecoming a specialist in bioinformatics

DNA fragments automatically. Looking back, this may have been thestarting point for me to pursue bioinformatics.
However, concepts such as "bioinformatics" didn't actually exist at thetime. My seniors and colleagues around me at the time really excelledin their fields, and it seemed like it would be difficult for me to survive
in the academic world even if I continued my doctoral course. Afterobtaining my master's degree, I decided to work at the Yamanashi
Agricultural Research Center which was in my hometown. I was incharge of soil research and development together with rice andvegetable cultivation. It wasn't a bad life, but I was also asking myself"Do you really want to do this for the rest of your life?” I got marriedaround that time, and my wife was financially independent. So, I plannedon getting my PhD in a field where I could combine both computer
analysis and biology. That was what I was really interested in.

While I am currently conducting research and development as abioinformatician, I was an experimental researcher when I was in
graduate school. I was a member of Professor Kanji Oyama's laboratory
at the Faculty of Agriculture, Kyoto University, and participated inresearch such as figuring out the entire mitochondrial genome DNA
sequence of a liverwort —the first to be determined in a plant. The
method of genome analysis we were using at the time was very
primitive and something you could hardly imagine nowadays. It
involved creating a genomic library of liverwort using E. coli, reading
the sequence using a radioisotope, and then manually connecting thefragments of the genome that had been read separately. I grew tiredof the manual work involved and started to use a 16-bit computer inthe lab instead. I wrote a program that made it possible to connect

Fortunately, I heard that Professor Toshimichi Ikemura of NIG was1looking for a student. When looking through the "Application Guidelines
for the 3-year Course Program" of the newly established Graduate
University for Advanced Studies, the word "Computer biology" cameup. When I saw this, I thought "this is what I'm looking for!” This wasin 1992. At that time, the Human Genome Project was in its golden age,and the advent of the sequencer made it possible to read nucleotide
sequences at a very high speed. I went straight to Professor Ikemura
to speak with him. In our talk, he said, "We are planning to analyze
the large-scale structure of the human genome," and this is whatmade me decide to apply for the entrance exam.

time and effort on for genome analysis. Liverwort retains the mostprimitive mechanisms of terrestrial plants and is the ideal material forstudying their morphology and evolution. It has a long history as a model
plant, and the whole chloroplast and mitochondrial genomes of plants
were first sequenced in liverwort. Despite the entire genome also being
analyzed, and an outline of the chromosome level results being reported
in 2007, a thorough analysis of the complete chromosome did not makesignificant progress at that time.
Under these circumstances, 39 international and domestic universities
including the National Institute of Genetics, Kinki University, Kobe
University, the National Institute for Basic Biology, Tohoku University,
the US Department of Energy, and Monash University in Australia,
created a consortium to shed light on the overall liverwort genome
structure, which was a significant project. I believe that this consortium
was assisted by the full-scale introduction of the next-generation
sequencer that can read DNA sequences at a very high speed andwith high precision at a relatively low cost, and the development of
the "long-read method" that made it possible to read DNA nucleotide
sequences longer than 10,000 bases in one go. My laboratory group was
responsible for using the NIG supercomputer to link fragmented genome
sequences accurately, and for constructing a database by predicting
genes.

I entered The Graduate University for Advanced Studies as a 3-year
course student and started work. The target of my research changed
drastically from liverworts to humans, but no matter the species, thegenetic information is written with four nucleotides. In that sense,
bioinformatics is a field that has much freedom. Professor Ikemura
conducted his research through both experiments and analysis. Whenchromosomes are stained, both dark and light bands are visible. It is
known that the dark areas are rich in GC content (GC-rich) and there
are many genes present. Professor Ikemura was working on a theory
that there was some kind of signal present in the boundary between
the bands.
I was one of the few people lucky enough to have access to ahigh-performance computer at the time, which cost around 30 million
yen. While researching the human genome, which was my assigned
work, I also started building a codon database, creating a program that
could automatically convert codons for amino acids, which differ slightly
depending on the species. Professor Ikemura was the person who
started to develop the codon database, but he became too busy withhis work on human genome analysis, so I picked up where he left off.
After finishing the database, I launched a webserver and made it
public through the internet. This was when the internet was just startingto gain traction, and there were only one or two hundred web servers inJapan including the one that I had made.
I received my PhD in 1996 and was employed as a bioinformatics
researcher at the Kazusa DNA Research Institute in Chiba Prefecture.
I joined the group that successfully sequenced the entire genome of
cyanobacteria, with the sequence being completed in the same year.
It was the world's first completely sequenced genome of a photosyntheticorganism, and also the fourth completely sequenced bacterial genome.
It was around this time that I started working on data analysis, and at thesame time, I became a member of the steering committee of the databank of NIG (DDBJ: DNA Data Bank of Japan). In 2001, we completed
the genome sequence analysis of Arabidopsis, a generic model ofhigher plants, and in 2008, NIG offered me a position as a professor. I
decided to accept the offer and return to NIG, my former home, in 2009.
DDBJ was the main work to which I was initially assigned, but I also
became responsible for international collaboration later down the line,
and we're now working in collaboration with NCBI (United States), EBI(EU), and Genome Bank (China). Of course, we are also continuing toconduct genome analyses of various biological species. The main
targets of analysis so far include liverwort, citrus, and the domestic cat.

The results of the analysis clarified that there are around 20,000 genes
and the number of transcription factors that work as switches for genes
is smaller than in other terrestrial plant species, but there is a set ofgenes that is the basis for all land organisms. Accordingly, there is
now information on the use of liverwort as the basis for research intoa broad range of terrestrial plants on evolution, gene structure,morphology, information transmission, and metabolic systems. The
paper was published in Cell, 2017 (*1).
I think that the image most people have of liverwort is just "a moss
growing in damp shady places.” For plant researchers, though, thecompletion of the entire genome database was a long-awaited andpriceless accomplishment.

Catalogue of a wide variety of citrus
genomes, including mandarin orange(Citrus unshiu)

Liverwort (Marchantia polymorpha) as
the basis of terrestrial plants

Shizuoka Prefecture, where NIG is located, is famous for its oranges
due to its mild climate. For this reason, the Institute of Fruit Tree and Tea

Liverwort is an organism that I have spent an enormous amount of2

Science of The National Agriculture and Food Research Organization(NARO) is located there. We started genome analysis of citrus species
after I was invited to work with Dr. Tokurou Shimizu of the Genome
Research Unit in the same institution. The aim of the research was not
to carry out a detailed analysis of the entire linear genome sequence
as in liverwort but to roughly analyze as many species of the citrus
genus as possible and make a catalogue of their genomes. While there
are more than 150 varieties available on the market throughout theworld, the genetic diversity was too broad and there were manycases where the parental relationships were unknown. It would be
extremely useful for breeding citrus species if we could figure out
their phylogenetic relationships.

The American Shorthair is well known throughout the world and isfrequently used in crossbreeding with other breeds such as the
Scottish Fold. In other words, there are many breeds of cats that are
genetically related to the American Shorthair, so the genome information
gathered can be used for a broad range of purposes, and it is expected
to contribute to research in veterinary medicine. Furthermore, theancestors of the American Shorthair are the cats that first landed on
the American continent, so the genome information is also important
for research into the evolution of the domestic cat.
The DNA was sequenced at the Kazusa DNA Research Institute. We
used the NIG supercomputer to conduct gene prediction and genestructure analysis based on the sequence data. The key to our success
was making effective use of the long read method—the same method
used for the analysis of liverwort, and we were able to analyze 19
chromosomes very accurately, at almost full length. The entire genomeof the American Shorthair comprises 2,493,141,643 base pairs, and23,119 genes were found (*3). Making full use of our groups' experienceand intuition, we were constantly posing questions to ourselves suchas "Isn't this gene too large?" and "Is there any possibility that wehave used a fragmented gene to make the prediction?" I think making
use of AI in these proofreading steps could be effective in the future.

Accordingly, Dr. Shimizu and his team extracted nuclear and mitochondrial
DNA targeting 269 varieties planted in their institute and sequenced
each fragment. After receiving the sequence data, we analyzed andcompared the gene structure data and the sequence polymorphisms indetail. As a result, the parents of 22 species, including Citrus unshiu,
were identified and the combination of seed parent line and pollen
parent line was also clarified. In addition, we were able to obtain
phylogenetic information on 45 native varieties, such as identificationof single parents, place of origin, and parent-offspring relationships.
The paper was published in 2017 (*2).

By comparing the sequences of the American Shorthair and theAbyssinian, we also found that they have different genomic structuresin some parts of their chromosomes. In the future, we can expect to gain
insight into the genes associated with hypertensive cardiomyopathy
common in the American Shorthair by comparing the genes of the two

In some phenotypes such as those that are associated with taste ordisease resistance, multiple genes are involved. Accordingly, a
database that can provide a comprehensive view of all genes is likely
to help produce varieties with both improved taste and better disease
resistance. We have already registered the raw and analyzed data in
DDBJ.

breeds in detail.
It is often not possible to publicly disclose the results obtained when
we collaborate with private companies, but Anicom was willing to release
the results to the public. The results were published in DDBJ in May
last year. While only the sequence data is available at the moment,
we intend to continue to gather and organize medical-related and otherinformation and link it to pathogenesis-related genes to make it easierfor veterinarians to use.

The sequence of the whole genome of
the American Shorthair—a popular housecat breed both inJapan and overseas
We analyzed the genome of the domestic cat because of my affection
for them, and because it is known that there are genetic diseases that
tend to only arise in certain breeds. However, research on the prevention
and treatment of these diseases hasn't made significant progress because
important genomic information hasn’t been available. The domestic
cat that we know today came about when humans domesticated wildcats and bred them as companion animals. Crossbreeding with wildcats can be seen in some breeds. The only breed with an analyzed
genome is the Abyssinian, which was done in the US in 2007. However,the data is not comprehensive. Since Abyssinians are a breed in whicha high degree of inbreeding has occurred, there were few genetic
markers (marker genes) that could be used to compare with otherbreeds. In addition, the entire genome was obtained by reading thesequence of some 4,500 separate fragments and simply connecting
them. There were also many unclear parts in the genome structure at
the chromosome level.

Collaborating with researchers, whether
from industry, government, or academia

Fortunately, we could undertake our domestic cat genome project incollaboration with three organizations that have been at the forefrontof research in this field: a well-known pet insurance company, Anicom
Insurance, Inc. (Anicom Specialty Medicinal Institute), the Kazusa
DNA Research Institute, and The Chinese University of Hong Kong.
The breed we selected to analyze was the American Shorthair (female).

We are conducting collaborative research on genome analysis for awide variety of biological species, at a broad range of scales and3

levels, with researchers regardless of whether they are in industry,
government, or academia. This is because there is not a sufficient
national budget for scientific research and there are not many
informaticians who can conduct large-scale genome analysis. My
research attitude of "emphasize sequence information regardless ofthe species to be analyzed" may also have something to do with it.

because of its prestige, but because they have professors in their mind
whom they want to work with.
In fact, the genome analysis of the domestic cat was a joint research
project organized by Dr. Matsumoto, who was employed at the Anicom
Specialty Medical Institute. I was a member of the screening committee
for his doctoral dissertation.

I will continue working in collaboration with various industries,
governments, and academic institutions. At present, COVID-19 mutations
are an emerging problem. My research group is contributing to society
by carrying out genome analyses of mutant strains of the virus
originating from those who have tested positive in Shizuoka Prefecture.

In the past, there were only a limited number of post-doctoral careers, but
now there is much more freedom. In particular, in the field of information
analysis, private companies are willing to employ data scientists, so
the work environment and salary are excellent. I think given the
chance, young researchers should experience a variety of different
environments, both in academia and in the private sector. However,
because informaticians are more likely to get employed by privatecompanies that have the appeal of better conditions, they tend to leaveacademia, which is an issue. If there continue to be only a limited numberof laboratory head (or similar) posts in academia, and no improvements
are made, it is difficult for me to ask students to remain in academia
considering their future.

Although it may not seem useful at first, I think genome analysis isalso a very important form of basic research. For example, genome
information from liverwort may not be directly linked to industrial
applications, but it is essential to understand basic mechanisms interrestrial plants. I often tell students in my lectures that when Heinrich
Hertz discovered the radio wave, he said, "This is totally useless."
However, without the radio wave, there would be no smartphones that
we use today. Even if it doesn't seem to be applicable, pursue what you
are interested in until you really understand it. This kind of attitude isimportant for researchers.

In Japan, the University of Tokyo and Tokyo Institute of Technology
are very well known for their bioinformatics research, but I would liketo take this opportunity to emphasize that it is possible to engage in avariety of research at the Graduate University for Advanced Studies
as well. The pandemic continues to take its course, but we can conduct
research even from home if we have a computer at hand, meaning there
is little to no impact on our work. For those who think "This is for me"
and would like to join us, please don't hesitate to knock on the door of
the National Institute of Genetics. People like me who are alreadyworking members of society but would like to study again are also
very welcome.

Currently, we are also working on "genetic diseases caused by genome
instability" as a collaborative research project. It's a fairly large project,and many genetic diseases are sporadic with very few patients. Many
people say that there is little information that can be obtained by reading
the entire genome in one patient, so it lacks meaning. However, I don't
believe this to be the case. If the entire genome is sequenced in detailand the data is gathered, I think important relationships regarding
pathology will appear in the future. If you want to read the sequenceof a genome, I think the most important thing to do is to sequence
everything properly and gather the data.

Interviewer:
Naoko Nishimura, Science writer

Bioinformatics at The Graduate University
for Advanced Studies

Photographer:
Mitsuhiko Kurusu, NIG ORD
July 2021

I believe today's students at The Graduate University for Advanced
Studies are exceptional compared to the times when I was a doctoral
student. Communication in English is considered a matter of course,
and I get the impression that many students are motivated to leave theirmark in the world of academia. Some graduates even get employed byprivate companies after they obtain their doctorate. Everyone has astrong sense of purpose, and they are coming to the university not just4

■ Links to databases■ MarpolBase: Genome Database for■ Cats-I: Cats' genome Informatics■ References*1---------■ Insights into Land Plant Evolution
Garnered from the Marchantia
polymorpha Genome.
Cell. 2017 Oct 5;171(2):287-304.e15.

*2---------■ Draft sequencing of the heterozygous
diploid genome of Satsuma (Citrus unshiu
Marc.) using a hybrid assembly approach.
Front Genet. 2017 Dec 5;8:180.
*3---------■ AnAms1.0: A high-quality
chromosome-scale assembly of a domestic
cat Felis catus of American Shorthair breed.
bioRxiv
doi: https://doi.org/10.1101/2020.05.19.103788