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Section 1 of 1
Town Hall: Advances in the Understanding of the Activity and Diversity of Nitrogen-Fixing Organisms in the Marine Environment
Please fill any part of the form below for QUESTIONNAIRE 1 "QUANTIFYING DIAZOTROPHS" (questions 1-6) or QUESTIONNAIRE 2 "15N2 RATE MEASUREMENTS" (questions 7-12) and join a small group discussion. Thank you!
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QUESTIONNAIRE I: QUANTIFYING DIAZOTROPHS
Our goal: (RT)-qPCR is currently the most widely available approach for quantifying abundances of diazotrophs and their transcriptional activity. We want to hear your thoughts as to how we can - or if we should - improve our current approaches.
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1. What do you think are the biggest limitations of quantitative PCR and Reverse transcription-qPCR in assessing diazotroph abundance and activity?
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There are no major limitations, these methods are fine.
These methods are too specialized and/or not available to me/my lab.
They don't accurately track gene copy and transcript abundances due to various method limitations.
Number of published primer sets is too limited.
Other…
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2. Are nifH transcript abundances expected to correlate with rates data?
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Yes
No
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or
add "Other"
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3. Why or why not? (Are nifH transcript abundances expected to correlate with rates data?)
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(0 points)
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4. In the case of very low and debatable N2 fixation rates, what is sufficient molecular evidence to provide proof of diazotrophic activity? Choose as many as applicable.
Question Type
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DNA sequence libraries showing nifH sequences.
Transcript libraries showing nifH sequences.
qPCR or RT-qPCR data.
Other…
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(0 points)
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5. If there was an updated standardized method for qPCR/RT-qPCR for diazotrophs, what aspect(s) are you most uncertain about and would like to make sure is explained in detail: (choose as many as applicable).
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Sample collection (including how much volume is needed, storage on the boat, cleaning procedures etc.)
Designing and validating your own primers and probes.
Preparing standards.
Other…
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6. Are there other methods that you believe are currently at a development state to serve as a stand-alone or complementary standard method for quantification of diazotrophs?
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QUESTIONNAIRE II: 15N2 RATE MEASUREMENTS
Our goal: Despite recent changes in methods to account for gas equilibration and potential NH4 contamination of stocks, the 15N2 tracer method, in one form or another, remains the most widely utilized method for quantifying rates of diazotrophic activity in the ocean. Our goal is develop recommendations for a standardized approach and we want to hear your thoughts as to how we can - or if we should - improve our current approaches.
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7. What version of the 15N2 tracer method does your group utilize?
Question Type
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Some version of original 'bubble' method
Some version of 'bubble release' method
Some version of the dissolution method
These methods are too specialized and/or not available to me/my lab.
None
Other…
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8. If you use the dissolution method, what do you think are the biggest limitations of the approach to accurately measure N2 fixation rates?
Question Type
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Accurate measurement of initial atom % of 15N in enriched seawater
Potential contamination during enrichment preparation
Measurement of the initial atom% of 15N of particulate N
Error in other metrics (e.g. particulate N or isotopic composition or reproducibility of enrichments)
Other…
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9. Do you present detection limits for your samples and if so what do you report?
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No
Yes, detection limits are calculated via_____ (type answer in "other" below):
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10. Do you follow routine protocols for timing of incubations across all field experiments, e.g. 24h dawn to dawn incubations?
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No
Yes, we____ (type answer in "other"):
Other…
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11. If there was an updated standardized method for the 15N2 tracer method, what aspect(s) are you most uncertain about and would like to make sure are explained in detail: (choose as many as applicable)
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Experimental setup: which bottles to use, how to incubate, how to filter
How to make enriched seawater
Standardized measurements for detection of the dissolved 15N/14N
Determining detection limits including via error propagation.
Other…
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12. Are there other methods that you believe are currently at a development state to serve as a stand-alone or complementary standard method for quantification of N2 fixation?
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QUESTIONNAIRE I: QUANTIFYING DIAZOTROPHS
1. What do you think are the biggest limitations of quantitative PCR and Reverse transcription-qPCR in assessing diazotroph abundance and activity?
Copy
No responses yet for this question.
2. Are nifH transcript abundances expected to correlate with rates data?
Copy
No responses yet for this question.
3. Why or why not? (Are nifH transcript abundances expected to correlate with rates data?)
Copy
No responses yet for this question.
4. In the case of very low and debatable N2 fixation rates, what is sufficient molecular evidence to provide proof of diazotrophic activity? Choose as many as applicable.
Copy
No responses yet for this question.
5. If there was an updated standardized method for qPCR/RT-qPCR for diazotrophs, what aspect(s) are you most uncertain about and would like to make sure is explained in detail: (choose as many as applicable).
Copy
No responses yet for this question.
6. Are there other methods that you believe are currently at a development state to serve as a stand-alone or complementary standard method for quantification of diazotrophs?
No responses yet for this question.
QUESTIONNAIRE II: 15N2 RATE MEASUREMENTS
7. What version of the 15N2 tracer method does your group utilize?
Copy
No responses yet for this question.
8. If you use the dissolution method, what do you think are the biggest limitations of the approach to accurately measure N2 fixation rates?
Copy
No responses yet for this question.
9. Do you present detection limits for your samples and if so what do you report?
Copy
No responses yet for this question.
10. Do you follow routine protocols for timing of incubations across all field experiments, e.g. 24h dawn to dawn incubations?
Copy
No responses yet for this question.
11. If there was an updated standardized method for the 15N2 tracer method, what aspect(s) are you most uncertain about and would like to make sure are explained in detail: (choose as many as applicable)
Copy
No responses yet for this question.
12. Are there other methods that you believe are currently at a development state to serve as a stand-alone or complementary standard method for quantification of N2 fixation?
No responses yet for this question.
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