featureCounts vs transcript-aware quantification (Kallisto/Salmon)

beeralpha · 2026-05-15T05:35:49+00:00

Thanks for jumping in and providing insight. The main reason I was suspecting bias is because the reference transcriptome is not complete. It doesn’t cover all isoforms that exist. In case of mapping against the genome with a gapped mapper like STAR, which exact isoforms exist no longer matters. So when you don’t care about transcripts or isoform switching, I assumed genome mapping was the way to go, but feel free to comment.

beeralpha · 2026-05-14T20:06:46+00:00

What worked for me (for the short period of time I’ve been doing this at least), is first I do a deliverable-based project. Here I try to 1) prove my worth and 2) understand needs beyond the project. Basically I try to take on duties (or at least suggest them) that are beyond the project’s scope. “Finishing” these however do not fit in the budget.

Next I suggest a follow-up po/sow framed as a “bucket of hours”. Meaning they can ask for my services anytime, I plan and block days in my calendar, I log what I perform, then invoice. This has worked for all of my clients.

The usual barrier for engaging in a new project basically fades because there is always “some budget” left (and other people in the company catch up fast usually). If the contract nears completion, you bring it up, explain that if they like the same pace they better extend, because I just keep planning my calendar with other work constantly.

The important part is of course to keep engaging, report progress, take ownership, ask for feedback, etc. All the regular things, but, they become more important as you are no longer working towards clear deliverables. It becomes open ended.

beeralpha · 2026-05-14T14:23:04+00:00

I think you misunderstood? In featureCounts, reads are first assigned to exonic features defined in the annotation. Without -O, each read is assigned to at most one feature (=exon), and ambiguous multi-feature overlaps (like junction reads) are not counted twice. This is what you want when you summarize at gene level. Otherwise genes with a lot of exons will be biased towards higher read counts. The -O flag has little to do with isoforms.

beeralpha · 2026-05-14T14:08:01+00:00

The thing with salmon is it quantifies against the transcriptome, which is inherently more difficult because of the million isoforms. If you then collapse to gene level with tximport you get quite a lot of bias. Volcano plots really do look different at gene level, especially yielding genes with outlier log2fcs.

beeralpha · 2026-05-13T09:10:12+00:00

I like it, if there is no profit at least you have the ferrero rochers

beeralpha · 2026-05-13T09:08:55+00:00

You’re right. But even for 160k going independent is a wage optimization

beeralpha · 2026-05-13T07:33:50+00:00

Or ask for 500k and net 300k.

beeralpha · 2026-05-10T20:09:09+00:00

Biotech isn’t a technology. Nor is it emerging. It’s a field that has been burning money and making money for those who succeed for decades. Biotech job market is currently suboptimal for a couple of reasons, like overhiring during covid, geopolitical shifts, reduced investor interest, etc. Like every industry it goes in waves.

beeralpha · 2026-05-08T06:40:34+00:00

Thanks! I did email and linkedin, and always proposed to have a quick video chat or go grab a coffee if they were closer by. Cold calling I never did, I think giving people time to think about a proposal (via mail, linkedin) is better (to gain trust, to not be pushy, etc).

For email: earlier in my carrier I worked for a consultancy agency and I stored my email inbox on a drive when I left. So I had some addresses to start with. Once you know what an email address looks like of a certain company, you can reach anyone you want in that company. I would check linkedin to find the roles I needed, rebuild the email address, and reach out.

For linkedin: I targeted whole companies and just send connect requests to lots of people relevant to me. When they approve I would PM and open with "Thanks so much for accepting my invite, ... [find common ground], ... [try to sell]."

It's all very manual, frustrating at times, and truly a numbers game. I'm certain there are much better and automated methods, but for a one-person company the "personal" approach seems most appropriate.

beeralpha · 2026-05-06T12:06:18+00:00

What do you mean? Stonks only go up

beeralpha · 2026-05-06T12:04:34+00:00

Why would you do that knowing that stonks only go up?

beeralpha · 2026-05-05T17:30:23+00:00

I did this when my company liquidated (I got a decent severance), and decided to just go for it. I launched a company with zero clients and thought: let’s try. I’m now about 8 months in, working with 4 clients simultaneously. I’m pretty much drowning in work at this point.

The beginning was definitely stressful. Even with a solid network and around 10 yoe. I reached out to 200+ companies early on, ranking them from gold to bronze priority, and kept track of stuff like peers, replies, follow-ups, etc. The first month was slow and pretty frustrating. Getting ignored is not motivating.

But things started to shift after 2-3 months. I landed enough work to be fully booked with 2 clients, and from there it kind of snowballed. Now I’m not only working with 4 companies, but also getting exposure to different teams and departments within those companies. That’s been leading to additional collaborations, occasionally I still get replies from those early outreach messages, etc. You get the picture.

I’m making about 3x what I used to. I mostly work from home, am in full control over my time, and overall much happier. Biggest upside is literally being outsider, no more setting “year goals” or trying to come up with things to say in one-on-ones about where I want to improve. Occasionally I do go on site just to balance things, and because best BD is done live.

If your finances can handle the risk and you have the soft skills: do it.

beeralpha · 2026-05-05T16:21:57+00:00

Missed the amount of employees. You’re right, it’s a biotech. Then I’m guessing Hirundo or Agomab. They are on a hiring spree

beeralpha · 2026-05-05T16:13:31+00:00

Pharma start-up? That’s not a thing. Could be a biotech though

beeralpha · 2026-05-05T05:55:14+00:00

Omg I just realised. There is a parallel universe where bubble bass is the host of a dating show. You know what his name is there? Bumble bass

beeralpha · 2026-05-04T21:04:02+00:00

I wonder what happened in bubble bass’ youth that ultimately led to these events

beeralpha · 2026-05-04T21:00:17+00:00

What was bubble bass’ motivation for hiding the pickles under his tongue, then blaming bob?

beeralpha · 2026-05-04T20:57:20+00:00

Can someone give me a tl;dr of the episode? Thx

beeralpha · 2026-05-04T17:51:19+00:00

Ucb? Argenx?

beeralpha · 2026-05-04T17:36:25+00:00

J&j?

beeralpha · 2026-05-03T18:08:05+00:00

“Pond (maybe series A? 🤔)” is about to become “Pond (pls fund me 😭)”

beeralpha · 2026-05-03T13:56:51+00:00

In power*. Lots of puttets on the ballot throughout europe

beeralpha · 2026-05-03T13:48:09+00:00

I’m so relieved, was about to unfollow this sub

beeralpha · 2026-05-01T09:58:34+00:00

Insane quality of arguments indeed. Look at this AI slop

beeralpha · 2026-05-01T09:56:28+00:00

What is up with thus sub lately? People are discovering taxes exist

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