准备oral的时候，通读了自己写的五份答卷。给老板的题目里有两道开放性题，觉得很想留下来。
初心啊，绝对不能忘的。

4. Read the attached paper “Strong inference” by John R. Platt (1964) and comment on your dissertation experiments in light of the ideas discussed in the section “A Yardstick of effectiveness”. How do your dissertation experiments fare regarding “The Question” described on page 352?

Answer:

An important lesson to my life is: do not easily trust some famous people’s quote, even that one is your idol. You may have no idea why or where they said those words, so that those famous people are always highly risky to be misunderstood by public. I recently started reading of Einstein’s biography by Isaacson. He had a famous saying, which I don’t agree with at all, “If we know what we were doing, it would not be called research, would it?” This sentence is more like a placebo to a depressed man who is totally lost in his work.

In my opinion, after 3 year growth of my PhD study, people who are doing research should have a clear mind on what they are doing. And more over, people should clearly understand WHY they are doing this. Taking my beginning motivation of taking PhD as a negative example: I remember when I first came back and talk with my advisor on my dissertation, I actually had no idea what things I want to do. I knew something about Next Generation Sequencing (NGS), and considered this will be a direction of future science. I told my advisor simply that I wished to do something with NGS then to do bioinformatics analyses on computer. At that time, I was 28, have worked a little out of the Ivory tower. A PhD degree, to me, is no more than a shining tag, which could prove the society that I am smart and valuable. I wished to learn some fancy techniques, become somewhat expert and get into an upper level of this cruel society. I clearly remembered that I was waiting for a question given from my advisor, and was getting ready to be a good employee of the lab to finish the task.

Fortunately and unfortunately, I got no specific task but a simply reply: you can do whatever you want. This is how my PhD education started, and which guide me to think about “questions” very first time. I can still remember the lost feeling of my first several weeks back to school. “Is this called research? I totally have no idea what kind of things should be researched, and totally have no idea what things I should do. ”

This was how my research first started. Luckily enough, my advisor did not just watch me drowning to die but offered several small projects as life jackets to keep me floating, moving and breathing. I was then coming back to complete a sheath nematode identification note, working on PCR of lance nematodes, anatomical observation of two lance nematodes, and finishing a class essay on phylogenetic analyses. That’s my first semester of my PhD student life. These guidance of small questions offered from my advisor finally helped me to build my own project.
I spent so many words on narrate a background information is because I think the step from zero to 1 is the most important step.

Once my brain started to move, several potential directions of my dissertation came out: to study biogeographic distribution of lance nematodes, to compare two species of lance nematodes, and to generate mitochondrial genome data of lance nematodes. Although there are several directions, they all belong to a big project: to collect information of lance nematodes and to build database for this increasingly risky plant-parasitic nematode. All of those questions could utilize NGS techniques, however, mitochondrial genome data could serve the other two directions as well., which is more fundamental. Logically, if we could generate more and accurate molecular data of lance nematode, both population genetics and parasitism function study would have more reference to stand on.

When I picked mitochondrial genome sequencing as my direction and presented it on department seminar, my sight on PhD study had changed a lot. As what I teach my students in TA now: never ignore some simple methods or techniques. Only if they could solve key questions, they could be the best techniques.
When my objective became clear and big question was targeted, I still met a huge number of questions in detail and got lost. Just like life that people always know and understand some big life principles, but always hard to achieve them in daily behavior, which is just the distance between a saint and a mortal. During my work on mitochondrial sequencing, I went through several steps and failures before I finally succeed. At the beginning, we simply want to get mitochondrial genome out. The technique difficulty was how could we get enough mitochondrial DNA from a single nematode. I tried a mitochondrial genome amplification kit, but failed many times. I tried Long PCR methods, failed many times again. I was totally lost in how to amplify mitochondrial genome based on poor amount of molecular references.
There is a key reason of my lost. I was trying to take advantage of NGS to generate data, but I didn’t follow the developing of this technique. NGS is developing all the time, and in 2016, it was already mature and capable to deal with single cell sequencing using whole genome sequencing. On the other side, although I expect the size of mitochondrial genome should be similar as other nematodes, this was only an unwritten hypothesis in my brain and I didn’t do anything to prove it. Moreover, the most important mistake I made: at that point I totally forgot my original purpose of building molecular database for lance nematode research, but just stuck in the failure of amplification of mitochondrial genome and became blind.

The light beam came into my darkness was my second committee meeting in November 2016. In the meeting, my committee members offered me two vital points to finally pull me out of the morass. One is on ”why do you want to sequence the mitochondrial genome”? The other one is on “why not try to amplify the whole genome and then pick out mitochondrial genome?” Although I kept trying another two month long PCR after the meeting, I finally notice one important thing: I am trying to eat a kiwi fruit in front of me, but my behavior is not like a human being, but like an ant. If the seed inside of kiwi fruit is the mitochondrial genome, as an ant, I can only dig a deep hole and try to reach it. If I am a human being, I will eat the entire the fruit, and I eat the seed as well. Especially, to use a spoon to peel the kiwi fruit is now a mature, systematic method. I started to refresh my mind and finally figure out a systematic way to obtain the mitochondrial genome: whole genome size estimation, theoretical coverage calculation of whole genome and mitochondrial genome, libraries preparation and sequencing, and assembling. Once you have a clear question, the really right question, you will have the best effectiveness on you research. If I didn’t grow up a little from the little ant, I may possibly get the kiwi seed after a huge amount of work. But eventually, the question is not only about mitochondrial genome, it’s all about lance nematode and their references, which is my original question for my PhD dissertation.

What Einstein said about research might be partially right: if we forget our purpose, we will get lost and have no idea what we are doing, which is normal in scientific researches because of the limitation of humanity. However, keep reviewing, thinking, and self-correcting is what we learned from science. Life can always find a way out and become better only if you keep fighting. I do love Einstein’s another saying, and strongly agree with it, ”Life is like riding a bicycle. To keep your balance, you must keep moving. ”