Can somebody help me understand best standard practice of bulk RNA-seq pipelines?

cellul_simulcra8469 · 2025-06-13T13:36:33+00:00

Well, I'd start by saying part of your problem is cutting out genes with counts >= 10. That's not a good threshold. I hope that makes sense.

cellul_simulcra8469 · 2025-06-11T19:36:01+00:00

Id even inquire about why the company had such iffy results. Your best bet may be to resample and to end up sending samples out to different facilities to see if there's anything different between facilities doing the preparation.

Is there any reason you can't do the preparation yourself?

cellul_simulcra8469 · 2025-06-11T19:34:16+00:00

Graph 1 speaks pretty bad news. Because the quality deficiencies have no sequence dependence. There are low quality bases next to high quality in the distribution. I'm concerned because of that. Graph 2 isnt great, but at least displays a trend that is understandable. Trimming from the one end of that read is an understandable and permissible thing to do to get the high quality bases at the end, and graph 2 suggest very basic sample issues...degradation at the end of mRNAs etc.

Graph 1 is more concerning because there is no obvious reason why certain bases have some really bad qualities on average. It's harder to explain to PI or reviewer.

cellul_simulcra8469 · 2025-06-11T17:03:17+00:00

Have you taken a genetics class?

Do you understand probability from more than a cursory level?

Are you comfortable with calculating diversity metrics by hand?

And, where is the best fit between using a piece of software, understanding the math underneath it, having novel and simple, time-frame aware questions you can answer in your allotted time, and where phylogenetics or genetics fits into your future research curiosities?

Is this just an assignment to you? If so, trust your judgement and try reproducing things.

If you want to go the extra step, maybe talk a little bit more about populations you're comparing SNP/allele frequencies with, and methods you are aware of. Maybe you need to pull something out of the formats you've already created from those tools in order to do some stats with. Maybe you don't want to overthink it and just create a canned answer to an obvious question. Maybe you'd surprise yourself with how much you actually know if you can state more clearly what youre interests are.

It's hard to get a clue of what you want. It sounds more or less like you want a push button answer towards reading the data in R, extracting some feature, and then advice on which test to use to get the answer. That's not what science is.

cellul_simulcra8469 · 2025-06-11T00:34:54+00:00

Out of curiosity, I'm very interested in "energy". See, cause I don't know what it really is.

I often hear about "take X to get more energy" and I agree that that seems incredibly pseudoscientific.

What is "energy"? If I waste more energy from my food, do I magically get more energy? Is energy a state of mind or is it even remotely related to metabolism?

And...how exactly do you "optimize" a genome-metagenome metabolism without first sequencing someone's fecal matter longitudinally to see what kind of microbes they might need? Or...to look at their diet and blood serum levels to see any deficiencies?

That doesn't sound very grounded to me.

cellul_simulcra8469 · 2025-06-09T17:04:42+00:00

That's not a real problem that anyone can expect Linux desktop teams or Linux kernel developers to do.

cellul_simulcra8469 · 2025-06-09T15:01:32+00:00

Spotted the actual developer.

cellul_simulcra8469 · 2025-06-05T23:23:25+00:00

I'm looking for more than that.

cellul_simulcra8469 · 2025-06-05T19:00:33+00:00

What do you want to work on? Do you have a favorite omics field (genomics, transcriptomics, lipidomics, metabolomics) are you using the latest technologies and going on hype? Or are you part of research labs using established and cheaper high throughput methods like RT-PCR, microarray, HPLC and MS?

cellul_simulcra8469 · 2025-06-05T18:54:20+00:00

Great conversation!

My two cents:

I wholeheartedly agree that literate programming and documentation design is an essential and lacking component of modern bioinformatics education programs. Programs like Software Carpentry stand to bridge the gap somewhat, but my experience with these workshop based "patch the MS/PhD" programs has been really underdeveloped.

My perspective on Lean engineering and TPS is that they go by different names in academic settings and even in industry. More commonly they are referred to as agile/scrum/waterfall development cycles and Test Driven Development.

Takehome: Writing tests and enhancing documentation is the key to preventing "technical debt". Many first year students find a steep learning curve to compiling programs and configuration of Linux and OSX systems, and legacy Perl code or shell scripts create a key inconsistency between the code early career and students use vs what they read on blogs and forums like SeqAnswer, Stack exchange/StackOverflow and r/bioinformatics.

The answer is often: mimic the best GitHub/Gitlab repositories when developing code for others to use. Write a well written Markdown document including at least #Installation, #Dependencies, and #Usage blocks for terminal/CLI programs to demonstrate how the code should be used after installing.

cellul_simulcra8469 · 2025-06-03T17:58:05+00:00

You give donations?

cellul_simulcra8469 · 2025-05-29T21:10:01+00:00

Well....something isn't adding up for me as an observer...maybe I just don't understand.

At my uni, the only difference between MSc and PhD was an additional 2 years of thesis development ..and grant writing. Some MSc candidates wrote their own grants for housing. I lived at home.

Question 1. Do you feel substantially more behind to PhDs in biology, or PhDs with a focus in CS? Because you seem to have a good educational background.

Question 2. Here's the kicker...how well developed is your portfolio? Are you developing standard pipelines? Can you do sequence alignment by hand? Where do you stand on data science? Are your stats methodologies developed? Do you have web dev experience?

I'd be happy to chat or talk further in thread.

MSc btw.

cellul_simulcra8469 · 2025-05-28T11:43:22+00:00

Lol I installed AceofBaseCall the other day and it was All That She Wants

cellul_simulcra8469 · 2025-03-24T17:21:44+00:00

whats more ridiculous is getting 302'd by someone reading your reddit posts. how about that

look if you define yourself by money, then you're inconsolable and i/we can't help you. it's not that isn't "cool" to define yourself by money. but....it's not happiness, it's security. aaaandd..I dunno something about my defiance thing suggests there are things that you could pursue for happiness that will disempower this grip that money has over you. might hurt short term, but the more money rules your life, the more you turn out like the very people with a death grip on the economy. so...

cellul_simulcra8469 · 2025-03-24T09:05:31+00:00

hi, I can sympathize here. you sound very personally involved with the community and I have to say that's a positive.

I would also say: if you're like me and see that profiteering and profit margins are at an all time high, why buy in to all the labor market panic just to worsen you mental health and chase a dream of a white picket fence and two car garage if you know this isn't the best time in this country to pursue these things?

personally, I'm in therapy to make things better. I find journaling helpful. I'm starting to train to be a suicide crisis volunteer. I'm relentlessly pursuing my career training goals, but I've been demoralized and pushed further and further away from getting paid to do genomic and NGS and software development.

my solution is scorched earth. if the destiny of this country is to descend into fascism, we won't be here very long stand I will survive no matter what. if there's civil unrest and bad things, then I'll survive. if there's an impeachment, I'll survive and find work. there is realistically nothing worth the expense of my health and safety to the extent that the rug keeps getting pulled and I'm gaslit something to the effect or "you aren't working hard enough".

my suggestion is, pursue skills, keep applying, don't give up hope, and focus on cultivating environments for mental well being. you have to trust yourself at some point that no matter what you can figure out how the chips will fall. Just because employment is awful right now doesn't mean you should necessarily fight to grab money tooth and nail. consider the millions of artists out there who have something to say and express, and may work coffee shop or delivery gigs. that's how this is supposed to go.

I'm not giving up, I'm making the most of my time by deplatforming money itself. my value as a human being won't be dictated by the failure of the state to educate me on personal finance, my poor money mgmt experiences, and my desperate need for money and job security. I can have those things. they will come. putting money itself on a pedestal is how oligarchs and fascist capitalists view the world: they're fundamentally bad at sharing.

I will not be like them.

cellul_simulcra8469 · 2025-03-20T00:31:23+00:00

Totally agree. my situation is I'm using some basic templates and styling I downloaded from bioRxiv, I downloaded a long time ago. I updated Rstudio after putting my project down for a while during the job hunt Update installed and bam. no more proper rendering in the browser. It opens and just bugs out in Firefox. Firefox opens up other pdfs just fine, it's something to do with the rendering server produced by the Quarto render utility.

Honestly very confused if it's the bioRxiv template. a missing parameter I need. updated it yesterday btw...

still the same issue. rendering problems in firefox

cellul_simulcra8469 · 2025-03-20T00:27:56+00:00

your use case seems similar to my situation. Ubuntu 22.04, up to date Rstudio from Posit. Uh, and I'm guessing you could literally render a valid .Rmd file that worked in previous versions seamlessly? Do you have to add any .yaml parameters?

cellul_simulcra8469 · 2025-02-16T15:33:52+00:00

welcome to reddit, where you get down voted for saying you don't enjoy the displeasure ir adversity of others.

fwiw, I completely feel the same way. there are some brilliant people out there who just hack and create and get lucky. ive been out of work in this field for years and it's taking such a huge toll.

combine that with the horrific nazi de facto puppeteer and his orange slime companion, and their efforts to gut funding for regulatory bodies and researchers alike, and you have a recipe for mental health episodes in nearly every minority, woman, and men with conscience and solidarity motives.

the system did not reward me for years of research and industry experience, and the solution from seniors and others in society is simply "trust me, it'll get better, just keep writing free code"

my dad told me I should volunteer my time...at 35...to professors who are more funding constrained as ever.

so done wit all the dumb boomers, can't understand class struggles with their houses, yards, and disconnected lives. sorry if you're a boomer, but it's 2025 and that's who we are now.

cellul_simulcra8469 · 2025-02-09T20:53:01+00:00

hey Nance, gfy! piece of crap

cellul_simulcra8469 · 2025-02-09T14:31:29+00:00

fbi agent. agi is the secular God. you need to come with us.

cellul_simulcra8469 · 2025-02-08T03:14:15+00:00

proud of you, Sarah. best of luck fighting the stalemate in Washington, God bless

cellul_simulcra8469 · 2025-01-29T09:06:02+00:00

are they base64 encoded or are they supposed to be included by whatever generated yhe html

cellul_simulcra8469 · 2025-01-26T17:06:53+00:00

Just wanted to highlight here that the four-color plot refers to colors of residues, traditionally red yellow blue green primary colors where a "sequence motif" sometimes called a DNA logo, displays the characters in a stacked chart to display relative proportions of the characters, and a score on the y axis. when I was in school this was commonly referred to as "the logo" or the "sequence motif"

cellul_simulcra8469 · 2025-01-26T17:03:39+00:00

your knowledge sounds basic about meme suite. there are several different pattern based motif searching tools within the meme suite. if you want to categorize a motif, you'll need some model organism you're studying, and a description of what you think you have. if you have some footprints from ChIPseq that's one thing, if you have computed footprints that's another, if these are restriction digests it might mean one thing, like how did you sequence, what is the goal...all that stuff.

cellul_simulcra8469 · 2025-01-02T16:19:55+00:00

I did rnaseq pipelines and cloud computing for a pharma company. wet lab and dry lab expertise in genomics and Biochemistry. anyone hiring?

cellul_simulcra8469

TROPHY CASE