我怎么能每天站在这大地上却感受不到它的力量？我怎么能站在这上面度过我的人生却不想知道它？— W. B. Logan1

    开始接触微生物之后，我发现我很喜欢这一类神奇的物种，关于土壤微生物组取样世界标准化这个想法已经酝酿三年多了，但依旧不成熟，自己水平有限，就放到这来留作纪念吧。非常欢迎各位老师同学来交流这个问题，本人的思考还具有局限性，另外可能很多类似的文章我没有发现，所以非常欢迎大家来探讨，在此感谢。

The storage temperature which have been descrbed in the published articles were -20℃ (Wang et al., 2018) and -80℃ (Knelman et al., 2012), and most of them were -80℃. As we know, this was the laboratory conditions, not the transportation conditions, but how was the influence of the transportation process to the changes of microorganisms? To be honest, the research field of most ecology researchers were far from their laboratories, so the temperature of -20℃ and -80℃ are difficult to achieve in the wild areas. So I have a question: Does the microbes in different soil typs around the world (Selcer, 2015) respond differently to different transportation or storage temperature?

In view of the above ideas, I designed two experiments. We chosed a maple forest growing for many years in our school as the research site. First experiment: "S" type sampling method was used to collect 10 pieces of soil in a 10×10 m plot with a diameter of 5 cm. The sampling depth was 0-10 cm, and the samples were mixed into one piece. At the same time, the samples were divided into 6 pieces and placed in 50 ml centrifuge tubes respectively. One was taken directly to the laboratory for DNA extraction, and the other five were placed in liquid nitrogen and refrigerators at -80℃, -40℃, -20℃ and 4℃, respectively. DNA was extracted after 3 days. Second experiment: The collected soil was directly brought back to the laboratory for soil DNA extraction, and then the extracted DNA was divided into 5 parts, which were placed in liquid nitrogen and refrigerators at -80℃, -40℃, -20℃ and 4℃respectively. Four days later, the samples of first and second experiments were sent to the sequencing company for high-throughput sequencing. Such a research and development of the small scale is somewhat like freezing and thawing test, and considering the mentor’s project did not involve the content, and this part down relative cost is higher, so I have temporarily not to do it, also had to wait after work to apply for the fund project to complete the study, but still have limitations of soil types, so I think whether can in the name of the journal to invite different regional researchers in soil microbial to submit manuscripts which could be as special issue to complete the study and sovle this problem.

      This year, I read two articles on methods, both of which were published on Nature Biotechnology. After reading them carefully, I felt that they were quite well done, but at the same time, new problems arose for me. As we can imagine, there are a lot of companies to do the high-throughput sequencing up to now, and there were all kinds types of DNA extraction kits, and there were definitely differences, but what are the differences, and what are the processes that make the difference, we found some answers in these two papers. Costea et al. (2017) evaluated the effects of 21 different DNA extraction methods on intestinal metagenomic results. He used two samples and then the samples are evenly divided into 21 pieces which come from 11 countries. The 21 laboratories used at least four different methods of DNA extraction for samples received. DNA obtained from 21 laboratories was sent to a sequencing center for database construction and sequencing (Hiseq2000, 3.8gb /Sample) to exclude the influence of experimental sequencing phase on the results. In conclusion, different methods of DNA extraction can significantly affect the extraction amount, integrity, intestinal flora abundance and diversity index of DNA samples. That was very interesting and the results are a good answer to the difference in different methods. However, there are two problems that bothered me. One was that is it necessary to send samples to different countries? Repeat the experiment in different laboratories at the same school may get similar results. Because, we couldn’t quantify the impact of transportation to different countries. The other one was that if we choosed the different companies may get different results.

 Sinha et al. (2017) evaluated the effects of different stages of 16S amplicon macrogenome sequencing (DNA extraction sample preservation sequencing and data analysis) on the results and found that different experimental stages (sample extraction, database construction, data analysis) have certain influence on the final results, among which DNA extraction has the greatest influence. To some extent, this paper supplemented the information not covered in the first paper, but also showed that DNA extraction had a great influence on the experimental results.

      On the one hand, more and more people begin to pay attention to the study of microorganisms, and the detection methods are emerging in an endless manner. However, there is no unified standard for the most basic part of sample collection and DNA extraction, so I am confused and worried. On the other hand, there's more and more research on amino sugars which as tracers for microbial necromass. In fact, we don't know much about the bacterial generation cycle or how environmental factors affect it, so the results were uncertain. Maybe what I said is not quite right, and I am also in the process of continuous learning, so I may not be very clear about many professional theoretical knowledge. I look forward to hearing from you . If you have any queries, please don’t hesitate to contact me at the address below.

Refrences:

Costea, P.I., Zeller, G., Sunagawa, S., Pelletier, E., Alberti, A., Levenez, F., Tramontano, M., Driessen, M., Hercog, R., Jung, F.E., Kultima, J.R., Hayward, M.R., Coelho, L.P., Allen-Vercoe, E., Bertrand, L., Blaut, M., Brown, J.R.M., Carton, T., Cools-Portier, S., Daigneault, M., Derrien, M., Druesne, A., de Vos, W.M., Finlay, B.B., Flint, H.J., Guarner, F., Hattori, M., Heilig, H., Luna, R.A., Vlieg, J.V.H., Junick, J., Klymiuk, I., Langella, P., Le Chatelier, E., Mai, V., Manichanh, C., Martin, J.C., Mery, C., Morita, H., O'Toole, P.W., Orvain, C., Patil, K.R., Penders, J., Persson, S., Pons, N., Popova, M., Salonen, A., Saulnier, D., Scott, K.P., Singh, B., Slezak, K., Veiga, P., Versalovic, J., Zhao, L.P., Zoetendal, E.G., Ehrlich, S.D., Dore, J., Bork, P., 2017. Towards standards for human fecal sample processing in metagenomic studies. Nature Biotechnology 35, 1069-+.

Knelman, J.E., Legg, T.M., O'Neill, S.P., Washenberger, C.L., Gonzalez, A., Cleveland, C.C., Nemergut, D.R., 2012. Bacterial community structure and function change in association with colonizer plants during early primary succession in a glacier forefield. Soil Biology & Biochemistry 46, 172-180.

Selcer, P., 2015. Fabricating Unity: The FAO-UNESCO Soil Map of the World. Historical Social Research-Historische Sozialforschung 40, 174-201.

Sinha, R., Abu-Ali, G., Vogtmann, E., Fodor, A.A., Ren, B.Y., Amir, A., Schwager, E., Crabtree, J., Ma, S.Y., Abnet, C.C., Knight, R., White, O., Huttenhower, C., Project, M.Q.C., 2017. Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium. Nature Biotechnology 35, 1077-+.

Wang, C.Y., Jiang, K., Zhou, J.W., Liu, J., Wu, B.D., 2018. Responses of soil N-fixing bacterial communities to redroot pigweed (Amaranthus retroflexus L.) invasion under Cu and Cd heavy metal soil pollution. Agriculture Ecosystems & Environment 267, 15-22.