Research for the higher-order brain mechanisms such as cognitive function and sociality from the perspective of molecular biology

Toru Takumi
Senior Team Leader, RIKEN Brain Science Institute
(The affiliation in the article is at the time of the interview.)

Our final goal is to understand the higher-order brain mechanisms such as cognitive function and sociality from the perspective of molecular biology. However, people cannot be approached directly. Therefore, we focus on the relationship between genetics and psychiatric disorders and aim to clarify the molecular mechanism of brain function by preparing genetically modified mouse models of human psychiatric disorders.

We are also establishing a cell model by introducing genetic mutations associated with psychiatric disorders into mouse ES cells. For rapid sequencing of DNA of vectors and ES cells, the SeqStudio Genetic Analyzer with its simple, all-in-one cartridge method is utilized.

Sequencer is just a tool for us, so we couldn't afford to put too much effort into it.
In this regard, the SeqStudio Genetic Analyzer is easy to use and convenient. We also like its speed, allowing us to obtain the results within a day, which is faster than outsourcing.

Genetic analysis of pediatric neurological disorders utilizing Sanger sequencing

Shinji Saitoh
Professor, Department of Pediatrics and Neonatology, Graduate School of Medical Sciences, Nagoya City University
(The affiliation in the article is at the time of the interview.)

We have worked on the genetic analysis of pediatric neurological disorders for a long time. In particular, we have identified causative genes of disorders of unknown cause that cannot be diagnosed (undiagnosed disorders) and disorders that are difficult to diagnose since they are rare disorders, and have worked on the elucidation of their underlying molecular mechanisms.

When academic theses are submitted it is necessary to not only confirm genetic mutations using NGS but also Sanger sequencing. Although whole exome sequencing is outsourced to an external institution, Sanger sequencing could rapidly be performed using the SeqStudio Genetic Analyzer that had newly been introduced at the laboratory.

Capillary replacement was difficult when using old model of the Genetic Analyzer and only a limited number of people could perform the replacement. However, anyone can perform replace now since the SeqStudio Genetic Analyzer is a cartridge-type. Our lab is a clinical laboratory and we often perform experiments in the intervals between providing services. Therefore, simplification of operations is of great benefit to us.

Elucidating the transcriptional regulation mechanism of ABO gene expression and its application to healthcare

Rie Sano
Associate Professor, Department of Legal Medicine, Graduate School of Medicine, Gunma University
(The affiliation in the article is at the time of the interview.)

ABO blood group typing has been used for blood transfusions, criminal investigations, etc. since its discovery in the early 20th century. The ABO blood group is determined by the ABO gene-encoding glycosyltransferase; however, the complete mechanism of ABO gene expression is not known. The focus of blood typing is a few antigens on the surface of red blood cells, and we aim to elucidate the transcriptional regulation mechanism behind it and its application to healthcare.

The unique mechanism of ABO blood typing has been an obstacle for research. For example, in case of BO type, it is necessary to accurately prove which allele has the gene mutation such as deletion. The same applies to sequence confirmation after genome editing. We have developed a method to specifically amplify only the A and B alleles of the ABO gene using PCR. Since Sanger sequencing is used for the sequence confirmation, it is still essential even now that next-generation sequencers are widely used.

Sequencing had been performed using the single-capillary ABI PRISMTM 310 Genetic Analyzer in the past, but we have now introduced the SeqStudio Genetic Analyzer. Previously, experience had been required to replace the capillaries and polymer; however, since SeqStudio Genetic Analyzer is a cartridge type, anyone can replace the capillaries and maintenance has become easier. Since an 8-tube strip used for PCR can be set as it is, it is also convenient from the perspective of the flow of experiments.

Development of basic technologies for genome editing and applications

Tetsushi Sakuma
Associate Professor, Graduate School of Integrated Sciences for Life, Hiroshima University

Genome editing that has been widely used as a tool for life science and has attracted attention from society. Together with Professor Takashi Yamamoto, the presiding chair of our laboratory, I am working on the development of basic technologies for genome editing and the applicable research involved in joint research.

In the development and application of new genome editing technologies, it is necessary to produce many plasmid vector constructs to use for genome editing. When producing complex constructs, the SeqStudio Genetic Analyzer is used at each step to check the sequences.

Since SeqStudio uses an all-in-one cartridge method, it does not require polymer replacement by experienced personnel, which makes maintenance much easier. In addition, since the equipment can be operated directly using only the touch screen without having to be connected to a PC, it is easy to teach students joining the laboratory every year how to operate it.

Identification of origin of bigeye tuna imported into Japan using genetic analysis of the mitochondrial DNA

Satoru Chiba
Tuna Fishery Resource Group, Tuna and Skipjack Resources Division, the National Research Institute of Far Seas Fisheries
(The affiliation in the article is at the time of the interview.)

Tuna is found in seas all over the world where it is caught/used by Japanese and foreign ships, and has to be protected and managed as an international fishery resource. A limit is therefore placed on the allowed amount of annual catches depending on the species and the location. As a responsible tuna-consuming country, Japan needs to prevent the import of tuna caught outside the international resource management framework. Especially concerning bigeye tuna, which accounts for the highest volume of tuna distributed in Japan, the National Research Institute of Far Seas Fisheries entrusted by the Ministry of Agriculture, Forestry and Fisheries, can identify the sea location where a bigeye tuna that has been imported into Japan was caught using genetic analysis.

Atlantic bigeye tuna and Indian Ocean/Pacific tuna can be distinguished based on genetic analysis of the mitochondrial DNA. Using this method, we randomly select and inspect about 150 vessels per year among overseas vessels without any advance notice, and investigate 25 samples per vessel. Genotypes are first analyzed using the Applied Biosystems™ TaqMan® assay, and those among them that cannot be identified are analyzed by sequencing. Since the distribution of fish is halted for the investigation, accuracy and speed are required of the analysis. TaqMan assay is helpful since samples received in the afternoon can be identified by the evening. If the sequence is also required later on, those results can be obtained the following day using the Applied Biosystems™ SeqStudio™ Genetic Analyzer.

Since the previously used analyzer was a single-capillary ABI PRISMTM 310 Genetic Analyzer, it took time to analyze multiple samples. However, since the number of capillaries has been increased to 4 and operability has improved, analysis can be performed within a short time without stress.

Utilizing SeqStudio in the core lab of the university for molecular biology research

Tsuyoshi Waku
Associate Professor, Genetic Code Laboratory, Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University
(The affiliation in the article is at the time of the interview.)

Our main research is about understanding the relationship between transcription abnormalities and protein degradation and cancer exacerbation.

Before Purchasing SeqStudio, outsourcing without having to own a sequencer has also been considered. However, after discussions with doctors of the research department, we concluded that as a research institution, we have to carry out the basic technique of molecular biology called sequencing at our own facility. As an educational institution, we also considered it important to show the actual instrument during lectures to undergraduate students. Therefore, we decided to adopt SeqStudio at Common Equipment Room of the Department of Medical Life Systems.

Since SeqStudio uses an all-in-one cartridge method, polymer replacement and buffer replenishment are unnecessary and maintenance time can be reduced. Due to the highly flexible opportunities to perform analysis and the short run time, analysis can be completed within the same day if samples are received in the morning. Since our previously used 3130xl Genetic Analyzer was a 16-capillary analyzer, it would sometimes take a long time to collect 16 samples, or we sometimes had to run the analyzer without filling all the spaces despite the high running cost. The SeqStudio Genetic Analyzer is a 4-capillary analyzer, so it can be run with 4 samples and used flexibly. As long as mass analysis is not necessary, small-scale analyzers can reduce the running cost and are suitable as jointly used equipment.

Moreover, in the past, the only way to deliver the analytical data was attachment to an e-mail. With the introduction of the SeqStudio Genetic Analyzer, data transfer through the cloud has become available. If the client wishes to receive data through the cloud, the analytical data is automatically saved on the cloud after completion of sequencing and is accessible from PCs at each laboratory. Even when the operator is absent or cannot send email due to other work, the client can still have immediate access to the analytical data.

Genotyping to ensure quality control of plant bioresources

Using SeqStudio Flex system’s flexible plate setting effectively

Ms. Atsuko Iuchi, Ms. Yukie Aso, Ms. Hiroko Saito
RIKEN BioResource Research Center Experimental Plant Division, Japan

RIKEN BioResource Center is a Japan’s leading establishment for the collection, storage, and distribution of bioresources. The Experimental Plant Division works with Arabidopsis thaliana (thale cress) and Brachypodium distachyon (purple false brome) seeds, as well as cultured cells and genetic materials of various plants. “We have 570 Arabidopsis thaliana wild seed lines and 250,000 full-length cDNA clones. We use genotyping to verify the exact line when distributing our resources to those who wish to use them or when running routine quality control checks,” Ms. Iuchi and her team explained. We asked them how they use the Applied Biosystems™ SeqStudio™ Flex genetic analyzer they recently adopted and how it differs from the Applied Biosystems™ 3130xl genetic analyzer that they have used previously.

Verifying plant seed lines through genotyping
“Up until now, we’d been using two 3130xl systems, but this model will no longer be supported at the end of 2022, we’ve recently adopted the SeqStudio Flex system for plant bioresource quality control. When it comes to Arabidopsis thaliana line genotyping, we run fragment analysis for 16 SSR sites. SSRs are simple sequence repeats, like STRs used in human identification. When we upgraded from the 3100 to the 3130xl system over ten years ago, we struggled with different electrophoresis patterns of fragments between the two models. This time around, we compared the new machine with the previous model once again. Even though the capillary length increased from 36 to 50 cm, the electrophoresis patterns were practically identical. It matched the previous data, which saved us a lot of trouble,” Ms. Iuchi and Ms. Saito said. Furthermore, “In bioresource work, we run QC on multiple samples, so installing the high-throughput SeqStudio Flex system has improved our productivity. And as we usually handle samples in units of 4, 6, or 8 in a sequence of quality check run, the 24- capillary system is in line with our workflow, allowing us to get accurate quality check results,” Ms. Saito continued.

Sequencing analysis with greater accuracy
“For cultured cells, we sequence specific regions of genes such as rbcL gene used in DNA barcoding once a year, both to ensure that there is nothing wrong with passaged cells and to verify whether we get the same results as the previous year. When we distribute genetic materials such as full-length cDNA clone of Arabidopsis thaliana, we confirm the sequence and supply　the data together with the material,” Ms. Aso said. “We also compared the system with the previous model in terms of sequencing, and although we only ran a few analyses, SeqStudio Flex system’s base calling showed improvement in distinctive sequences such as polyC sequence with continuous C’s and polyC sequence followed by another continuous sequence. We have experienced miscalls with the previous system due to successive peaks not separated clearly, so we had to visually check the data again in such cases. In contrast, the new system demonstrates improved peak separation, enabling automatic base calling. Such highly objective data also provides reassurance as we supply the data,” she continued.

Flexible plate setting
Lastly, we asked them what they like about the SeqStudio Flex system the most. “In the past, we had to check with other staff members in advance and get all samples in order before starting a run. But with the new system, even after someone starts a run, we can add sample plates at any time. We can, for example, perform fragment analysis followed by sequencing, or we can swap the order of plates when we need to reprioritize samples. Such flexibility is a great advantage. By performing routine QC testing and on-demand testing needed prior to supplying our materials in seamless manners, we hope to see our productivity improve,” they said.