2025 is gone, bringing with it another opportunity to share some highlights from a warehouse worker’s swing at studying our living history a sheaf of genome graphs and shelves full of protein-forms at a time.

Last year was both series of great adventures and turnabouts recorded in incomplete pieces of notes and impressions strewn out through the months. I think this is a good time to finally pastiche together those scraps into a travelogue of the year – maybe there’s another independent researcher out there someday who might find these experiences interesting.

Here are the two major highlights of the year.

ASM Microbe 2025: June 19-23

Last thing I remember before waking up was gently rocking heave-ho of the massive diesel train slowly pulling itself out from among the labyrinthine tunnels beneath white marble slabs of the new Moynihan hall, all grinding sound of metals and chains echoing through the dark.

Storm swept summer Kansas plains rolled by in a vivid haze of great horizontal lines outside the window – gold, brown and green under permanent grey cast of clouds trailing a sizeable tornado across the state. I didn’t expect what should have been a monotonous scene to be so overpowering, showering us with scent of the storm and grass tamed by the train’s HVAC. Unexpected transition of the train into a tornado chaser, however, meant our route into Colorado was blocked with precariously swinging power lines and trees strewn about, leading to ample delay (of about 24 hours) and chances to enjoy gamut of conversations among misfits in the cabins consisting of school teachers, truckers, civil engineers and a big group of Amish travelers that later broke out into songs over a bit of accordion. It was a welcome reprieve from gawking at shifting ecotopes and struggling to make my way through the Notebook of Malte Laurids Brigge, which was somehow proving to be a denser read than the Magic Mountain at less than half the volume.

Unexpectedly, these conversations proved to be a high point of the journey (I also had a lot of fun cactus-sighting during the Arizona leg of the trip – turns out we were just a bit too north for a good Saguaro spotting). There’s nothing quite like hearing about the Colorado River Compact from two retired & retiring civil engineering veterans from Nevada and California sitting across a table by pure happenstance, excitedly discussing current state of the Hoover dam reservoir like some erstwhile actors from the set of Chinatown. The delve into the unique bit of Americana was punctuated by moments of occasional silence from the gentleman from California, turning his coffee mug while staring into it with creased eyebrows – I believe he was en route to meet his son from Mexico.

These random chats gave me much needed opportunity to share my research to series of fresh audience as well, held captive by Amtrak steak dinner and desserts. A more memorable impromptu presentation of my ASM 2026 poster was with a trucker couple from Illinois – proudly having traveled to the 49 states (sans Hawaii) on their own wheels, they were taking it easy aboard a train for this journey. After a roller-coaster of threads ranging from their support for rounding up illegal immigrants, questions about latest covid conspiracy theories and difference between Archaea and Bacteria, I gave a somewhat condensed version of the poster-side chat with some neat finger drawings.

This one's more of a small demo of the Archaeal sequencing and analysis data I've been building up since last year - covering the whole Halococcus genus! 

All out of pocket, lifting boxes and selling bagels out of an aluminum box working at my own converted warehouse lab. It's looking like a genome library for a whole evolutionary branch of Archaea is set to balloon from a warehouse lab contribution by the end of the year.

You see, last year an academic partner and I found that a species of Halococcus, H. dombrowskii, carries an rrn operon on both its chromosome and plasmid (an rrn operon is just an operational unit for expressing ribosome - it's a protein we use as a signpost to figure out how related one species is to another - for now). This is an expansion of the study to see if the chromosome-plasmid distribution repeats across the entire genus or if it's specific to ours. 

On top of that, I'm gathering additional data on methylation state of the new genomes while sequencing them (methylation's a chemical modification on the DNA itself, decides whether the thing actually does something or not), since it's supported out of the box with latest flowcells. These genome sequencers are about yea big - measured in inches and fits in a pocket. Darn expensive to run though. It looks like methylation pattern on the chromosomal rrn differs from that found on the plasmid, which I'm really excited about.   

It sounded like they were quite enthused by the aspect of a warehouse worker’s presentation at an academic conference (maybe more so than the study itself) – “go get’em tiger” they said, among other encouraging things.

Abstract:

Euryarchaeon Halococcus dombrowskii has an unusual genomic architecture with multiple rrn (ribosomal RNA) operons distributed across its chromosome and plasmids. Our previous study on differences between H. dombrowskii’s plasmid and chromosome rrn operon ITS (Internal Transcribed Spacer) sites suggested there could be further quantitative differences among these disparate rrn operon regions. As the rrn operon ITS regions in question occur close to a well-characterized tRNA site known to interact with archaeal RNase, we performed an exploratory methylation survey of the H. dombrowskii genome to check for genomic compartment-specific rrn operon methylation patterns and site differences.

Here, we present preliminary long-read based direct methylation calling data for H. dombrowskii ATCC BAA-364, describing a genome rich in N4-methylcytosine (4mC) followed by N6-methyladenine (6mA). Initial survey describes a minor methylation site difference between regions in and around chromosomal and plasmid rrn operons that will require further analysis. We also note a curious lack of N4-methyltransferase annotation for the H. dombrowskii genome, and offer a putative candidate gene for further study based on HMM profiling and sequence homology analysis.

We hope to utilize the data and tools we developed for this initial study on follow-up long-read sequencing and direct methylation calling analysis of other Halococcus spp. Additional data would allow us to perform vitally needed comparative genomics analysis, providing us with further fascinating insight into archaeal ribogenesis in genomes with multi-compartment rrn operon placement.

Poster available at:

https://doi.org/10.5281/zenodo.16369513

After a quick engine failure at the entrance of the Mojave desert and some jumping wire fences and buses, I finally made it to LA in time for last tram into the city – at the strike of midnight before beginning of the conference.

Conference experience is far too long and complex to go through here – I was certainly happy to see familiar faces, many of them even welcoming. Quite a bit of valuable input on how to shape the Halococcus data as well. The discussion on pangenomic approach is well reflected in a preprint I’m writing up at this moment, set for final QC and submission this Spring.

I even got to rerun the poster spiel with more than a few people. One postdoc responded with her mouth agape in horror when I got to the warehouse and bagel part – “Oh I’m sorry!”

Boston Bacterial Meeting 2025: June 09-10

This one was on a whim. My first complete genome sequencing project after buying the MinION (there were other incomplete ones before) was that of Deinococcus radiophilus ATCC 27603 undertaken with another amateur biologist friend in 2019.

In retrospect, the sequencing was the straightforward part. Considering we had to spend months to scrounge up our own HMW genome extraction method from scratch in the dark era of ONT R9 chemistry, this is saying something. Beloved Flye was still in beta stages, and state of polishing tools were nowhere near what we have now. For moderately complex genomes, complete de novo assembly of acceptable quality just wasn’t very achievable up until Trycycler and Homopolish came on the scene (the latter’s replaced by many maintained alternatives, and Trycycler is superseded by Autocycler in most scenarios now – tooling’s come a long way).

It bears repeating – anything worthwhile in independent and amateur research would not have been possible without open source software and accessible hardware.

Figuring out genomic compartment level completeness of an assembly without a reference was pretty tricky to figure out in the beginning – and I had to learn about alignment, tree building and comparative genomics methods quite quickly. And then a method to track presence of highly variable genes across different genomes – in my case a task I had to learn basics of HMM and HMMER suite for. Tearing apart and studying well documented pipelines like GToTree was essential for this part. I still recommend GToTree (https://doi.org/10.1093/bioinformatics/btz188) and its documentation as a standout example of what an open source research software documentation should be.

At the time, studying completeness of a Deinococcus radiophilus genome meant understanding how other Deinococcus genomes are structured, and that eventually led to an interesting observation. It turns out vast majority of species in the Deinococcota phylum maintains large (100kb+) plasmids driven by a specific plasmid replication initiator protein (RPA) belonging to a single protein family with shared motifs and domains, representing close to 90% of the known genomes in the group (the percentage’s been steadily climbing as more genomes within the phylum are sequenced). So – for whatever the reason, the phylum Deinococcota represents the highest concentration of RPA proteins and RPA protein driven plasmids among 19 bacterial phyla surveyed from NCBI collection, spanning about 11k QC’d and deduplicated genomes (these numbers are significantly higher now – 11k genomes are from 2019 when I was just starting to learn about API access and other bioinformatics things).

The RPA proteins are always a component of an operon next to parA and/or parB genes, with recognizable palindromic sequence motif following the operon – so chances of the HMM screening results being entirely false positives is unlikely.

While further study of RPA distribution and evolution in nature was held off back in 2019 due to life circumstances, I thought it was about time I do a proper study of RPA evolution within the Deinococcota phylum. I wanted to further pursue my initial idea that unusually high concentration of RPA among Deinococcota is because they’re distributed through inheritance from an ancient common ancestor.

And what better way to present this work-in-progress than an early career/student researcher facing conference like Boston Bacterial Meeting?

Abstract:

Replication initiator protein A (RPA) under family PF10134 are often found in bacterial genomes as the main component for specific types of single strand DNA binding plasmid replication initiator sites, alongside accessory partitioning proteins A and B.

Screening 11082 reference bacterial genomes representing 19 phyla for RPA plasmid replication initiator protein (PF10134) and accessory partitioning genes show drastically uneven distribution of RPA plasmid operon in nature, with phylum Deinococcota hosting an exceptionally high concentration of the mechanism at 89% of member microbes representing extremes of habitat heterogeneity.

Multiple alignment and inferred tree experiments using Deinococcota RPA proteins show patterns mirroring rooted whole genome single copy gene (SCG) phylogenetic trees of their hosts. Current data suggests wide distribution of RPA based plasmid replication system exclusive among members of Deinococcota is based primarily on inheritance rather than introgression. Further study into evolution of RPA plasmids in Deinococcota could provide us with an unusual model system for studying plasmid evolution at scale.

Poster available at:

https://doi.org/10.5281/zenodo.16369073

The folks at BBM also offers scholarship to offset the registration costs – and fortunately thought my application was interesting enough for attendance. Much thanks to the nod, I honestly would not have been able to attend this one without the waiver.

The meeting was a fruitful one – multiple attendees suggested that I focus on analyzing the nature of conserved domains and motifs within the Deinococcota RPA proteins before proceeding further (I was originally considering expanding the screening outside the phylum to see if there’s an ‘ingress point’ protein out there somewhere). Some new tools and databases were suggested as well, something I’ll address in a separate note addressing this study.

What surprised me about the meeting was an atmosphere of universal focus on trimming down the research question until an observation is a wetlab problem. This Deinococcus study, for example, would not be considered fully featured (publication worthy) unless I started doing transformation characterization of the RPA protein and some model plasmid carrying it. I mentioned that I’m not sure if the approach is within the purview of a study on potential RPA common ancestor for a phylum level group – but was left with an impression that a strictly tree building study would be lacking.

I think that’s a fully justifiable view – but how would one design a wetlab counterpart to a study like this? Understanding the RPA on a more mechanical level should provide the answer, and is currently my focus on trimming and redirecting this study.

A memorable session at the conference was an elective titled ‘Science for the Public Good.’ I wasn’t quite sure what to expect wading my way through the posters and crowds, only to find the classroom packed with audience with an almost palpable sense of curiosity and excitement in the air. As an outsider the choice of most hotly debated topic during the session remains curious to me (graduate student unionization), especially within the context of public good. Even within the scope of graduate student well-being, isn’t it natural to also consider separating the identity of the scientist from their institutional affiliations in pursuit of agenda separate from those of the funding sources and funding administrators?

I got the sense that contemporary academia in the US largely considers itself under siege from what they consider outsider elements, such as the US government. However, is this really an accurate perspective when we consider the model of post-WWII scientific research built up under leadership of people like Vannevar Bush that we still operate under – where science IS the government/university complex? Again, it was fascinating to observe a cultural gap like this firsthand – this is something I’m aiming to address fully sometime in 2026.

Another notable experience was running into some people from SeqCoast sequencing company. Inexplicably, multiple greetings and questions to one of their senior personnel were met with pointed silence and staring off into the thin air. It’s been a while since I’ve seen someone try to ignore someone else with such intense effort. Although one of them came up later for a chat, past years of identifying myself as an independent researcher taught me to give a wide berth with organizations like this. What an odd experience, one would think people would be more talkative at academic conferences.

2025 in numbers

This post/note was originally a lot longer – addressing some of the extremely disheartening, personally damaging experiences with academia this year as well as my thoughts on requirements & attitude for an independent researcher that began to emerge throughout the year. However, those thoughts deserve a more complete, separate coverage.

It would be best to close out the last day of 2025 looking back on the more hopeful things, of solid discoveries of previously unknown things in this great, beautiful universe.

I genuinely believe there is a room for the identity of the scientist separate from other social institutions, and that everyone with the passion and drive can try their hand at scientific discovery in the same way being a poet or a novelist is not tied to our social backgrounds and affiliations. It’s just the question of building the way.

Happy 2026 everyone, the year of the Fire Horse!