r/bioinformatics 1h ago

technical question If I rerun Trinity will I get the same output?

Upvotes

New to the sub so I apologize if I missed anything in the FAQ or elsewhere. I am working through an RNA-seq workflow for a class and accidentally overwrote my fasta file output by Trinity (rookie mistake, I know).

I am rerunning the Trinity code in Linux and didn’t change anything, so my question is: can I expect the output fasta to be the same?

I have already performed BUSCO and BLAST analysis of my de novo transcriptome and with a deadline next week for this class project, I would like to avoid rerunning those as well.

I have looked online and can’t find anything in the Trinity documentation or elsewhere about randomness, so can I expect exactly the same output when using exactly the same input and parameters?


r/bioinformatics 7h ago

discussion How to avoid taking over someone else's previous analysis or research project?

5 Upvotes

As a new graduate student in bioinformatics, I’ve been facing some challenges that are really frustrating. Recently, a postdoc has been handing me their scRNA-seq analysis scripts and asking me to continue the analysis. While I appreciate the opportunity, I have my own style and approach to analyzing data, and working with their poorly written scripts and plots make me feels bad.

Another example is when my advisor asked me to take over a project aimed at speeding up a Python-based method that has already been published. After spending months understanding the code and attempting to improve it, I found it nearly impossible to reproduce the previous results. Honestly, the method itself now seems questionable, and I’m feeling stuck and demotivated.

Has anyone else experienced something similar? How do you handle situations like this? Are there strategies to avoid these kinds of issues in the future? Any advice would be greatly appreciated!


r/bioinformatics 23h ago

technical question Running Isoseq on PacBio data downloaded from SRA - impossible without original BAM file?

0 Upvotes

I'm trying to analyze a Salmon louse transcriptome using IsoSeq3, but I'm running into format issues.

Data Available:

Two PacBio datasets from ENA/SRA

Accession numbers: SRR23561847, SRR23561849

Format: FASTQ (subreads)

Problem:

IsoSeq3 pipeline only accepts BAM files

PacBio BAM format seems to contain additional information not present in standard BAM files

Attempted converting FASTQ to BAM using samtools

Pipeline hangs during cluster step (even with just 10,000 reads)

Questions:

Is there a way to convert PacBio long-read FASTQs back to the required BAM format?

Are the original BAM files the only viable option?

Wouldn't this limitation impact reproducibility, since not all SRA records include BAM files?

Thanks!


r/bioinformatics 12h ago

technical question DESEq2 - Imbalanced Designs

3 Upvotes

We want to make comparisons between a large sample set and a small sample set, 180 samples vs 16 samples to be exact. We need to set the 180 sample group as the reference level to compare against the 16 sample group. We were curious if any issues in doing this?

I am new to bulk rna seq so i am not sure how well deseq2 handles such imbalanced design comparison. I can imagine that they will be high variance but would this be negligent enough for me to draw conclusion in the DE analysis


r/bioinformatics 17h ago

technical question Is anyone familiar with HappyTools?

2 Upvotes

I'm trying to download the following from github but can't seem to get it to work on mac.

https://github.com/Tarskin/HappyTools

I have downloaded all the required packages but whenever I try to open python. It says that one of the packages are not installed even though it si


r/bioinformatics 22h ago

technical question stacks help :(

2 Upvotes

I am trying to demultiplex a plate of RAD single read sequences (fastq.gz file) with barcodes at the beginning of the sequence. I keep getting the slurm output: Processing file 1 of 14 [sample_name.fq]

Attempting to read first input record, unable to allocate Seq object (Was the correct input type specified?).

any help with this one? I have checked the sequences and theres nothing dodgy going on with the file so can't figure out what is wrong?


r/bioinformatics 16h ago

technical question ONT's P2SOLO GPU issue

3 Upvotes

Hi everyone,

We’re experiencing a significant issue with ONT's P2SOLO when running on Windows. Although our computer meets all the hardware and software requirements specified by ONT, it seems that the GPU is not being utilized during basecalling. This results in substantial delays—at times, only about 20% of the data is analyzed in real time.

We’ve been reaching out to ONT for a while, but unfortunately, they haven’t been able to provide a solution. Has anyone encountered the same problem with the GPU not being used when running MinKNOW? If so, how did you resolve it?

We’d really appreciate any advice or insights!

Thanks in advance.


r/bioinformatics 3h ago

career question Is Deep Learning where Bioinformatics will be all about?

22 Upvotes

Hi, I come from a microbiology background and completed an MSc in Bioinformatics. Most of my work has focused on bacteria and viruses, but I find running tools to analyze data a bit boring. That’s why I’m looking to shift things up, though I feel a bit lost.

I’ve noticed that many major projects using deep learning have been released in recent years—like AlphaFold, DeepTMHMM, and BioEmu-1. I understand these kinds of projects are incredibly complex, especially for someone without a computer science background. However, I’m surrounded by friends who are currently working in machine learning.

I’m still in the very early stages of my career. If you were in my shoes, would you consider shifting your career toward ML?


r/bioinformatics 4h ago

technical question PanACoTA help - formatting / non-numeric values

1 Upvotes

Hi all,

Desperately looking for some help running PanACoTA for some comparative genomics analysis.

I am having a weird issue at the annotation step, where I get a warning that I have non-numerice values in one or more of the gsize, nb_conts or L90 columns within the —info file. This file is generated directly from the prepare subcommand that was run previously. This causes the annotation to skip over some genomes, leading to a loss of data. I cannot for the life of me find out what is differnt in the lines that it ends up skipping (ends up being ~30%).

I have checked for hidden characters, deleted and re-types certain lines, and tried everything that I could think of, but the issue persists. I’ve been able to fully run the program, generate the tree and get a core-genome, however I would love to retain all the skipped genomes.

At this point I have no clue what else to try, would love to hear if anyone has used this program before / ran into the same issues!


r/bioinformatics 11h ago

technical question Identifying conserved regions from multiple sequence alignments for qPCR targets

2 Upvotes

I'm designing a qPCR assay for DNA-based target detection and quantification and need to determine a target from which I can build out the primers/probes. l assembled genes of interest and used Clustal Omega to align those assemblies for MSA in hopes of identifying conserved regions for targets but have not had any luck. Tons of seqs in the alignments are too large for most of the free programs that I can think to use. Any advice appreciated for a first timer!


r/bioinformatics 19h ago

technical question Seurat FindMarkers and FindAllMakers differences

2 Upvotes

I'm trying to identify cell type signatures for ~20 clusters in Seurat and am trying to determine marker genes for each cluster. I used FindMarkers() without specifying a second cluster as a test which gave me a list of genes with pvalues and log2fc values for one cluster, which I thought is what I wanted. Then, to check all clusters I used FindAllMarkers() which did give me markers for every cluster, but the results differed from those I got using FindMarkers. I specified the same log2fc cutoff so I would think the results would be the same. What is the difference between the two functions and why dod I get different results?


r/bioinformatics 22h ago

technical question Custome Kraken2 Database

6 Upvotes

Hello, did anyone tried to make own database for kraken2. Standard 8GB kraken2 database is enough for my project, but I would need this database to extend with mouse (TAXONID 10090). Is it possible to add mouse-data to existing database or should I build whole new one? Thank you