Ⅲ-4. Radiation Chemistry & Biology
RIKEN
Accel. Prog. Rep. 56 (2023)

RIKEN Accel. Prog. Rep. 56 (2023)

Semi-automated high-throughput microbial DNA extraction protocol
for population-scale mutation analysis
Y. Nishimiya,∗1 N. Lei,∗1 and H. Ichida∗1
At RIKEN Nishina Center for Accelerator-BasedScience, one active area of research is the application of heavy-ion beams to mutation breeding, where
we utilise next-generation sequencing and the massively parallel supercomputer “HOKUSAI” to characterise irradiation-induced DNA damage and subsequent genetic mutations statistically. In recent years,
we have demonstrated the eﬃcacy thereof using various model organisms, eﬃcient identiﬁcation of mutations through whole-genome and exome sequencing in Nicotiana tabacum L., and the applicabilityof the redundant sequence elimination algorithm inanalysing data obtained from a complex genome suchas wheat. We accordingly developed a versatile andhigh-accuracy mutation-detection pipeline.
Historically, only indirect measurements such as thesurvival rate and albino emergence rate were available to estimate the mutation-induction eﬃciency approximately. Whole-genome sequencing (WGS), on
the other hand, enables one to count the exact number of mutations in each sample, which presents two
crucial advantages: (i) The smaller error margin enables a more sensitive diﬀerentiation of the mutationinduction eﬃciency. (ii) Signiﬁcantly fewer samples arerequired to estimate the optimal experimental condition compared to conventional indirect methods.
Therefore, we began testing the WGS-based
mutation-induction optimisation methodology against
the survival-rate-based estimates using budding yeast
with a small genome size. However, investigating themultitude of irradiation conditions (ion type, linear energy transfer, and irradiation dose) and genetic backgrounds (wild type and DNA-repair deﬁcient mutants),
where all combinations thereof require several (at least10) samples, necessitates the simultaneous cultivationof cultures on a considerable scale whilst maintaining
the overall pace of execution to control for their timesensitive nature properly.
To overcome this challenge, we devised a series oftechniques to achieve eﬃciency based on the knowledge that 500 µL of culture per sample is suﬃcientfor DNA extraction and sequencing. This small volume requirement permits us to use 96 deep-well plateswith signiﬁcantly high density (a desktop incubator
can cultivate up to 384 samples) and higher ease ofmanipulation than conventional test tubes. Moreover, after centrifugation and a brief lysis procedure,
we can directly transfer them onto the automatedliquid-handling robot (OT-2, Opentrons) in a smooth
production-line-like manner. Eliminating the necessity∗1RIKEN Nishina Center- 195 -to pipette the liquid culture from one tube to anotherensures that the entire volume of the obtained culture
proceeds for DNA extraction, reducing human labourand risks of cross-contamination. The 96 deep-wellplates we tested can also be used for cryogenic storageof the liquid culture.
Hence, we tested the yeast DNA extraction throughthe automated procedure. Following trial and error, weveriﬁed the robot’s reliability to automatically executea protocol consisting of (i) cell-wall digestion with Zymolyase, (ii) extraction of DNA using magnetic beads,and (iii) elution of the extracted DNA. Although thereagents must be carefully prepared in advance, weestimate that the combined use of 96 deep-well plate
cultivation and robot DNA extraction would requireas little human labour as approximately half an hourin all to prepare up to 192 DNA samples per day, incomparison with the 4.5 hours including active andstandby time required for the manual alternative. Figure 1 presents an example of concentrations in ng/µLof the DNA thereby extracted across the well plate.
From past trials, we know that 700 ng is adequate
for sequencing. Thus, when the ﬁnal elution volume
is speciﬁed as 100 µL, the minimum concentration of
7 ng/µL suﬃces.

Fig. 1. Example distribution of extracted DNA concentration in ng/µL on a grid corresponding to the 96 deepwell plate. The blue-red bi-colour scale represents theincreasing yield.

At the time of writing this manuscript, we had acquired approximately 700 distinct yeast DNA samples following irradiation with carbon, argon, and ironbeams, the WGS data of which are presently being
analysed. Our current objectives are to establish thepopulation-scale WGS-based mutation analysis protocol as a more eﬃcient and accurate method to advanceour understanding of heavy-ion beam-induced mutations, DNA repair mechanisms, and their applications
to mutation breeding technology, and to develop statistical models to eﬃciently learn about and infer fromthe newly acquired rich source of genomic data.