# Note on extractions to NYU

Hi folks

We met extensively with Detlef this morning. In short, there are some significant discussions to be had about extraction methods going forward, but we agreed that we should not change protocol within a season. So, regardless of what we agree for next year, we agreed to continue with the bead-based extraction protocol we have used so far for all Fall 2021/Spring 2022 season samples from both FRA & GER.

Unless you have strong and immediate objections, we'll resume this work tomorrow for plant samples at least. We can discuss further tomorrow, but we think we can also proceed with membrane and companion plants using the same protocol. And given Luke's results on soil are very promising, we can probably go ahead with the soil samples too.

As we are still unsure if the plant (hamPCR) and other sample types (normal amplicon seq) will be procesed at the same time through the same library prep/ampicon method, we would still prefer to at least keep the plant samples in separate plates to avoid errors while "cherry-picking" the plant samples out of comingled plates.

Best,
Team Tübingen

# Pathcom Dilemmas

## RNA/DNA extraction methodology

- DNA and RNA in one protocol
- Which protocol is best for both? Econospin?
- Do we do everything with that protocol, including DNA-only samples?
- Soil: are we fine with the same protocol? @Luke

## HAMPCR

- plant = "Just" needs protocol optimisation & learning
- membrane = `????`
- soil = `??????????????????`
- companion = how universal is the GIGANTEA primer set? Will it work for non-Arabidopsis spp.?

## Trial/proofing of collection = minipathocom

- We need to generate an entire "mini-Pathocom" consisting of all data types from a few sites as a priority
- Most lab procols we can assume to work, at least with some optimisation or discussion
- Need to see experimentally if e.g. washing protocol is behaving as expected, if plants are surviving washing with reasonably intact endophyte microbiomes, if HamPCR works in all cases we expect, and ideally if we can extract RNA from these plants.

# scraps

In recent discussions, the Tübingen and NYU teams have found a dilemma regarding DNA extraction. In short, we face two opposing fears: On one hand, we fear that the economical protocol we have for DNA extraction will work poorly (and non-uniformly) across the various sample types, especially soil. On the other hand, we fear that the "kitome" batch effect between different DNA extraction methods would confound our results. Obviously, if we have a DNA extration method that for example only works for some soil types, that will lead to "kitome"-like biases, so these fears aren't mutally exclusive.

Short of extensive experimentation, does the group have any direct experience or suggestions?

**DRAFT: Follow up to USA team after Monthly with Detlef**

Hi all,

@Luke, thanks for updating us about the PCR results for the soil test. It is good to know the protocol works given the wet griding is well done.

From our side, also an update following our meeting today with Detlef.

**1. Mini DNA Pathocom.** Like we mentioned in the meeting yesterday, we will probably gain a lot by generating an entire dataset consisting of all data types from i.e., 3 sites. We will make it our priority for September and October so that we can have answers (if e.g. washing protocol is behaving as expected, if plants are surviving washing with reasonably intact endophyte microbiomes, if hamPCR works as we expect, and ideally if we can extract RNA from these plants)

**2. What protocol to use to extract RNA and DNA from the plant samples?**

Given that the purpose is to correlate microbial colonization with gene expression, ideally, we should use the same plant material for both. So, in the next weeks we will perform tests to also extract RNA as originally planned, using the same buffer from the current protocol, also test how it preforms when adding “RNA later” and compare all that with the Econospin protocol.

**3. What samples to use for hamPCR?**

The consensus is that hamPCR would only make sense for the endophyte samples (“plant” samples). This means extracting DNA from the plant samples on separate plates would be advantageous to directly process that for hamPCR. The other samples types - companion plants, soil and membranes - would only be used for the standard amplicon sequencing.

On that note we will finish the plant samples we already started because its half way through the season and it wouldn’t make sense to stop in the middle and just change things. To make things comparable, ideally, the US samples should also use the current protocol.

In the case we do change protocols (based on topic 2 above) we should only do it from the next season onwards.

By the way, according to the proposal, only a subset of total collected samples would be used for RNA-seq, so the Fall21 and Spring22 samples will not be included, so even if we change protocols later to adapt for RNA extraction there is no harm having these samples processed with the current protocol.

**4. Sampling Fall22**. We would like to suggested the rotation of at least 2 team members from each country to be a standard throughout the seasons and we detailed the idea in the Pathocom page. Considering there is only 2 months before the next sampling season, we really should start organizing this. What do you think about this?

Best,

# Bacterial WGS

- Nanopore vs HiFi vs illumina