First results - Revision history

Rachel: /* Recommendation + next steps */

2023-02-12T12:04:05Z

Recommendation + next steps

2023-02-12T11:59:37Z

People

2023-02-09T14:52:49Z

Further Feedback

2023-02-08T18:24:00Z

Recommendation + next steps

2023-02-08T18:21:27Z

Further Feedback

2023-02-08T17:07:19Z

2023-02-08T17:04:53Z

trying to get ITS4 to stay on right side!

2023-02-06T15:28:33Z

2023-02-06T15:23:06Z

2023-02-06T15:22:18Z

Further Feedback

@@ Line 172: / Line 172: @@
 We really would like to get back into the metagenomics barcoding, to see all the mycelia in samples, as in OpenFoodRepo DNA...
-We need to figure out some way to do this again.
+We needed to figure out some way to do this again, so are trying another [https://experiment.com/projects/participatory-research-to-explore-fungal-biodiversity-and-its-importance-to-bees special crowdfunding campaign], while moving forward as possible.
 Categories: [[:Category:Work In Progress|Work In Progress]]

@@ Line 3: / Line 3: @@
 === People ===
-Contact and lead:  Yngvar
+Contact and lead:  [[User:Cramy|Yngvar]]
-Participants: Rachel, other Hackuarium members, maybe *you*?
+Participants: [[User:Rachel|Rachel]], other Hackuarium members, maybe *you*?
 === Objective: ===

@@ Line 133: / Line 133: @@
 It was fun to see the hair ice (or something like it) just grow in our freezer (from a wet stick in a plastic zip bag).  We still have more sticks from Yngvar's colleague. However, we got samples to amplify by PCR from a sort of goo that grew on the stick, not from the ice itself.
-In the end the sequence from this culture gave ''Exidia thuretiana'' as its strongest sequence hit - with 105 bp identical
+In the end, the sequence from this culture gave ''Exidia thuretiana'' as its strongest sequence hit (using the classic analysis tool, known as BLAST) - with 105 bp identical
 ?? something close to the expected fungus ''Exidiopsis effusa''
-this still needs to be repeated.
+This still needs to be repeated, under better conditions for the 'hair ice' to really grow and be isolated...
 ==== A third objective for our experiments was to determine more about species that are not well known. ====
@@ Line 158: / Line 158: @@
   - just one hit and only 83% identity
-We have more more to learn and do
+We have more more to learn and do...
 == Further Feedback ==
-Trying to get samples amplified in the field (iYngvar in Italy) and sending for seq was a mess...  Eurofins did not get the DHL package even! (came back here weeks later) Needs more work.
+Trying to get samples amplified in the field (by Yngvar in Italy) and sending for seq was a mess...  Eurofins did not get the DHL package even! (came back here weeks later)
 == Recommendation + next steps ==

@@ Line 168: / Line 168: @@
 (We need high fidelity polymerase however, or the few key base changes that might occur with ordinary Taq would not provide reliable IDs...)
-Yngvar really wants to get into the metagenomics as in OpenFoodRepo DNA...
+We really would like to get back into the metagenomics barcoding, to see all the mycelia in samples, as in OpenFoodRepo DNA...
 We need to figure out some way to do this again.

@@ Line 161: / Line 161: @@
 == Further Feedback ==
-Trying to get samples amplified in the field and sending for seq was a mess...  Needs more work.
+Trying to get samples amplified in the field (iYngvar in Italy) and sending for seq was a mess...  Eurofins did not get the DHL package even! (came back here weeks later) Needs more work.
 == Recommendation + next steps ==

@@ Line 9: / Line 9: @@
 === Objective: ===
-==== To see whether species determination by simple PCR then sequencing, as we have already done for bacteria (with the universal 16S primers), will be possible. ====
+==== To see whether species determination by simple PCR then sequencing will be possible. ====
 Yngvar found some references describing protocols with ribosomal DNA 'internal transcribed spacer' (ITS) sequences that can be used as 'bar-coding' primers for fungus, in particular, a protocol from the Kennedy Lab by L. Higgins (2012).
 Unfortunately, this reference did not have the actual primer sequences in it, although it had a great overview image of the ribosomal DNA, which we reproduce roughly here:
 <big>'''ITS1'''**>...........................................................................................................................................</big>
@@ Line 21: / Line 21: @@
 To note, ribosomal genes come in big repeated clusters in most organisms, and this is the general layout of one of the standard repeats.
 When we got down to the sequence  level, in various references, we realised that some published sequences were not correctly transcribed, or at least, the ITS1 primer was missing a T in a few of the references (even in a PCR Protocol book from Cold Spring Harbor, put out in 1990).

@@ Line 14: / Line 14: @@
 Unfortunately, this reference did not have the actual primer sequences in it, although it had a great overview image of the ribosomal DNA, which we reproduce roughly here:
-<big>'''ITS1'''**></big>
+<big>'''ITS1'''**>...........................................................................................................................................</big>
 <big><18S rDNA | 1st Internal Transcribed Spacer | 5.8S rDNA | 2nd Internal Transcribed Spacer | 28S rDNA></big>
-<big><**'''ITS4'''</big>
+<big>......................................................................................................................................................<**'''ITS4'''</big>
 To note, ribosomal genes come in big repeated clusters in most organisms, and this is the general layout of one of the standard repeats.

← Older revision		Revision as of 15:28, 6 February 2023
Line 36:		Line 36:
	The first test of these primers was performed in our lab on February 3 2022, and we made an [https://www.evernote.com/shard/s586/sh/3cf6ffd6-fddc-d0e5-1faa-bec7d8e24d1b/36a5c24ba03e88f3bcde5b49e7cd4d27 evernote protocol with details]about comparing fresh, freeze/thawed and simple 'touch with a toothpick' comparisons.		The first test of these primers was performed in our lab on February 3 2022, and we made an [https://www.evernote.com/shard/s586/sh/3cf6ffd6-fddc-d0e5-1faa-bec7d8e24d1b/36a5c24ba03e88f3bcde5b49e7cd4d27 evernote protocol with details]about comparing fresh, freeze/thawed and simple 'touch with a toothpick' comparisons.

−	For one mycelia type, shiitake, all three reactions worked well, for another, only after the freeze-thaw was good, for instance. Ultimately we got sequences for all three samples, as shown below:	+	For one mycelia type, shiitake, all three reactions worked well, for another, only after the freeze-thaw was good, for instance.
		+
		+	After good PCR products are seen, it is still no guarantee of a perfect sequence, as a few differences can define the species. Generally, however, one good band would be purified with Qiagen columns and sent out for sequencing (Eurofins LightRun, or Microsynth seqs have been used over the years - also for bacteria as in our Montreux water work, with 16S primers...)
		+
		+	Ultimately we got sequences for all three samples, as shown below:
		+

← Older revision		Revision as of 15:23, 6 February 2023
Line 164:		Line 164:

	We need to figure out some way to do this again.		We need to figure out some way to do this again.
		+
		+
		+	Categories: [[:Category:Work In Progress\|Work In Progress]]