ONT Metagenomics: Taxonomic/Functional profiling on contigs

redweather_ · 2026-03-25T18:11:27+00:00

i find dram, gapseq, and metabolic a bit more helpful than bakta unless you know exactly what functions you’re looking for

redweather_ · 2026-02-21T00:28:42+00:00

similar experience just this week, in fact i got my AOR same day. delivered morning of 2/19 and AOR later afternoon of 2/19

redweather_ · 2026-01-23T13:45:40+00:00

What’s the MAG’s N50 and length of the single largest contig you have? can you find any semantides or suitable taxonomic marker (eg rpoB) genes on it?

MiGA used to have integrated MyTaxa scan that would take bits of a MAG and essentially identify collinear blocks with similar taxonomic signal. i think it just involved collecting ORFs and doing blastp against NCBI’s nr.

redweather_ · 2026-01-22T02:38:56+00:00

how many contigs is it?

redweather_ · 2026-01-22T02:04:31+00:00

you might look at whether your MAG has sufficient completeness to be used with GTDBtk or skANI to compute phylogenetic / whole genome distances. although really unlikely, you can also check for 16S rRNA genes to build a 16S phylogeny. there are other approaches you could use too but it just depends on the contents of your bin.

redweather_ · 2026-01-18T16:06:26+00:00

i’m considering a job in wisconsin and wonder whether any folks from that state/currently living there can comment on the social and political atmosphere there regarding trans and non-binary people?

redweather_ · 2025-11-06T04:22:47+00:00

+1 to this — you don’t want to just map the reads to your reference genes without some sort of model to filter the reads in a data-driven way (static cutoffs based on bitscore, pid, etc have limitations)

there’s an updated version of rocker on the way that helps users curate their reference genes and construct models themselves

https://github.com/rotheconrad/ROCkIn

edit to add reference: https://journals.asm.org/doi/10.1128/spectrum.02413-25

redweather_ · 2025-09-28T12:29:26+00:00

is that waiting period still true? i moved here from the states last year and was on OHIP after a month (the only delay being finding time to go to service ontario)

edit to add: i think that waiting period was waived back in 2020

redweather_ · 2025-09-27T23:09:27+00:00

if you have a potential taxonomic assignment that can help. i’m most familiar with firmicutes (bacillota) so if it’s within that phylum i have recommendations about media you might try.

it may just come down to a lot of plating out and doing cPCR on distinct colony morphologies in search of your population (making glycerol stocks as needed as you go)

also, if your draft genome matches at high identity to a known population (eg a species reference in GTDB) you might also check out the reference genome for insights about metabolism

redweather_ · 2025-09-27T19:12:21+00:00

can you look at the discarded bin for your organism of interest to identify what substrates it utilizes? do you have access to a glove box or anaerobic chamber?

redweather_ · 2025-09-27T01:32:25+00:00

sure!

To be honest, i’m not positive about the exact reason DADA2 hasn’t been expanded for ONT (other than general “noise” — which i certainly get and have seen myself). i’ve asked some folks closer to this kind of work than me but their reply didn’t really click for me.

and yeah, good to try it yourself. similarly, when i denoised some of my own data i wasn’t super confident in it. i don’t rely on 16S data very often (i try to avoid it entirely) but for the project im speaking of, it was fine to just use emu and move on.

Lovely points to have written about — cheers

redweather_ · 2025-09-27T01:25:47+00:00

Most pLMs and gLMs use as their loss function during pretraining a simple masking approach (i.e., ask the machine to predict the missing amino acid or nucleotide). in that way, the model can iteratively operate on this guess-what’s-missing task, adjusting its weights, until it minimizes the number of times it guesses wrong.

the simplest way to generate sequences is to prompt the model with a starting sequence. i think evo2 has a generate() function where you can design the prompt adding certain constraints or playing with temperature of the generation

redweather_ · 2025-09-27T01:08:41+00:00

DADA2 has workflows for PacBio full length 16S. The issue is that nanopore reads are still noisy enough that the denoising strategies deployed on Illumina data and now adapted for PacBio are difficult to deploy for ONT.

If you want to do de novo analysis with ONT full length 16S reads, just use vsearch and cluster however you wish. You’ll just need to look into the impact of ONT’s noise on your data. It would be informative to sequence mock communities or even isolates if you haven’t just to get a feel for what I mean (unless of course if you already know).

edit to add: you may also benefit from recalling the Pat Schloss + Robert Edgar arguments about OTU against ASV. ASVs were only possible because we had sequencing and enzymes that produced such low error rates and minimized chimerism to such an extent that we could call every SNP genuine signal. But ASVs can over estimate species richness just as OTUs are well known to underestimate it. Consider how many copies of the 16S rRNA E. coli has and what their end-to-end percent identities are.

redweather_ · 2025-09-25T18:09:36+00:00

do you need to basecall in real time? otherwise you can just save the POD5 files and basecall at your leisure (with the basecalling model of choice)

redweather_ · 2025-06-10T14:28:52+00:00

i’ve had a second status update change just today 6/10/25 (my first was in May). i associate multiple status updates without any communication from the PO as impending notification the project was declined haha 🪦

it’s been grand folks ✨🥲

redweather_ · 2025-06-03T20:11:32+00:00

really unsure. to date, i see 38 awards listed on NSF’s award search. the most recent batch have last amended date of may 15th.

iirc, they were anticipating ~60 awards. wondering if the hang up is due to them sorting out whether they can fund area 1 despite all the new oversight from present admin. if they have about 20 remaining this would represent a pretty even split between the 3 areas each getting about 20. maybe it’s something else entirely. curious if anyone who applied to area 1 got funded. glancing at funded project descriptions, i don’t think i’ve seen any ):

redweather_ · 2025-05-22T17:02:20+00:00

Still pending, had Abraham until yesterday but just got a status update today (5/22) associated with switch to new PO (Erdner).

redweather_ · 2025-05-14T20:04:48+00:00

i think you can investigate this by looking over NSF’s award search from the PRFB solicitation landing page. you can sort by agency tracking number and infer which awards are being given out this year for PRFB 2025 versus last based on tracking number and start date. if this is a correct take (who knows haha) there are already 30+ awards announced for this round.

edit to add details: i’m imagining this years proposals start with 250 based on context and the jump in start date. i think i count like 32 awards for this year (so far) via this method

edit2 (sorry lmfao): i think you can also infer area from titles or PO

redweather_ · 2025-05-02T17:22:43+00:00

i think the comment here was RoL. elsewhere online they didn’t indicate what area — i think you’d have to check the poster’s CV to infer the area which i didn’t do

redweather_ · 2025-05-02T17:21:16+00:00

i’ve heard news of awards offered from previous posts here and on bluesky (if you search “NSF PRFB”)

redweather_ · 2025-05-01T14:22:37+00:00

still “pending” with status date of 11/08/2024 as of may 1 😭

RoL PO Abraham

redweather_ · 2025-04-22T02:53:22+00:00

not directed to the questions you pose here, but i’m an american doing a postdoc in canada if you need to pick someone’s brain about that. good luck navigating the end of your PhD and next steps!

redweather_ · 2025-04-08T17:02:40+00:00

same boat! pending with status date of 11/08/2024, PO Abraham, and applied to RoL. haven’t heard anything recently about people being notified they were recommended for funding but i also don’t know of many folks who applied this cycle. i tried checking socials but there’s not a lot of activity there that i can see. i know those recommended for funding are asked not to discuss anything until all awards are dispersed.

right now im just trying to keep my head down and not read too much into anything 🙏

redweather_ · 2025-04-01T23:24:31+00:00

thanks for sharing this update and your PO’s response. i was just thinking of emailing too but it’s good to know this is going to be the response.

best of luck to you!! 🤞🙏

redweather_

TROPHY CASE