Bioinformatician are not anti-social; We are just genome friendly.

Bioinformatician would love to change the biological world, but they won't give us the genetic code :P

If at first you don't succeed; call it version 1.0

The glass is neither half-full nor half-empty: it's actually have several genomes.

I'm BioGeek.

Fedup with LIPS, try God script.

Idiot, Go ahead, make my data!

Thank god, my genome just compiled.

Error message: "Out of space on genome drive:"

Shut up mobile elements, or i'll flush you out.

Never underestimate the internet bandwidth, u gotta incomplete.

Applied fuzzy logic to understand God's logic?

Warning! Overflow, delete chromosome !

Be nice to the BioGeek, for all you know they might be the next curator!

Beware of computational biologist they screw genes and protein.

Warning! Your genome is full of garbage, delete it !

Bad or missing mouse genome. Spank the cat? (Y/N)

Genome make very fast, very accurate mistakes.

Let's BLAST it.

Some genome never has transposons. It just develops random features.

Go watch CINEMA and have BLAST.

The "Ifs" of NGS QC and Trimming

1. Ensures Data Integrity
   If you want to minimize errors in downstream analyses, QC and trimming remove low-quality reads and bases, ensuring high-confidence data. This step is essential for reliable variant calling, assembly, and other applications.

2. Removes Contaminants
   If adapter sequences or contaminants are present in the raw reads, trimming can eliminate them. This prevents issues like misalignment or incorrect biological interpretations, ensuring cleaner data for analysis.

3. Improves Mapping and Assembly
   If your goal is better alignment to a reference genome or improved de novo assembly, trimming low-quality bases and adapters is critical. High-quality reads map more efficiently and generate more accurate assemblies.

4. Reduces Computational Load
   If you want to save computational resources, trimming reduces the dataset size, which speeds up processing and analysis. Clean datasets mean less computational time spent on processing low-quality data.

5. Prepares for Standardized Analyses
   If your project involves multiple datasets, QC and trimming ensure uniformity across them. This standardization makes comparisons valid and reproducible, particularly in large collaborative studies.

The "Buts" of NGS QC and Trimming

1. Risk of Over-Trimming
   But excessive trimming can lead to the loss of informative sequences, reducing read depth and potentially discarding biologically relevant data. This is especially critical in studies with limited sequencing depth.

2. Bias Introduction
   But trimming algorithms might introduce biases, especially if they inadvertently remove sequences with specific biological patterns. This can skew results and compromise biological insights.

3. Loss of Context in Paired-End Reads
   But trimming one read in a pair more than the other can lead to loss of pairing information. This complicates downstream analyses that rely on paired-end data, such as structural variant detection.

4. Time and Resource Intensive
   But running QC and trimming for large datasets can be computationally expensive and time-consuming. As sequencing depth increases, preprocessing becomes a bottleneck in the analysis pipeline.

5. Variable Standards
   But the criteria for trimming (e.g., quality threshold, minimum read length) can vary between tools and datasets. This variability may affect reproducibility and comparability of results across studies.

Balancing the "Ifs" and "Buts"

To maximize the benefits of QC and trimming while mitigating the challenges, consider the following best practices:

• Use QC Tools Wisely: Start with tools like FastQC to identify quality issues in your raw data. Visualizing quality metrics helps tailor your trimming parameters.

• Choose Reliable Trimming Tools: Tools like Trimmomatic, Cutadapt, and BBduk offer adaptive and customizable trimming options. Select one that aligns with your dataset and project goals.

• Set Reasonable Parameters: Avoid over-trimming by setting quality thresholds and minimum read lengths that balance data retention and quality improvement.

• Test Downstream Effects: Validate the impact of QC and trimming on downstream analyses, such as alignment efficiency, variant calling accuracy, or assembly quality.

• Document Your Workflow: Maintain detailed records of the parameters and tools used for QC and trimming. This ensures reproducibility and enables better troubleshooting.

Conclusion

NGS quality control and trimming are essential steps to ensure reliable and accurate data for analysis. While the "ifs" highlight the clear benefits of these steps, the "buts" remind us of the potential pitfalls. By adopting best practices and carefully balancing these considerations, you can optimize your preprocessing workflow and unlock the full potential of your sequencing data.

1. Update new things continuously
As per my personal experience, it’s not always easy to work as a bioinformatician!  There are couple of reasons to say that; First computational part of biology make our life’s a little harder compared to other professional categories. The fact - for instance - that the technology cycle in the bioinformatics world is very short, the actual knowledge becomes outdated in a few months or years. Therefore, we need to learn continuously - new things get important. Second, to stay on top of things we really need the strong will to be good at our job. That's probably the most important characteristic to bioinformatician. They are usually an excellent knowledge worker with great technical abilities, and have the will to be that over decades!

2. Avoid the sentence "I did not know what to do!"
In our computational biology lab, we generally face lots of technical problems. But as you know, it's impossible to know everything to do the computational biology jobs ( Yup.. because you need diverse and multidisciplinary knowledge to understand biological problems and resolve their respective solutions), therefore it's absolutely necessary that a bioinformatician finds its way through a new topic. How I typically do that is I use google and I talk to other experts in our laboratory or online biostar community to find out what they think. "I did not know what to do!" should not be an argument for us.

3. To make oneself useful
Several time it does happen, you finished our task earlier than expected; in such cases if you have some time left then: Take a coffee and play chess; reversi, etc. In my case I take a rest. Afterwards I think about what I could do that helps the team to achieve its targets, 'cause some of my team mates probably didn't finish! (at least if I didn't met them at coffee bar !!)

4. Care for all
During my rigorous research duration; I attended several workshop organized by my University departments. I had a discussion with other research fellow, professors; I generally ask … what it really takes to make a team successful or to be a successful research leader. They always said: "Well, you need some caring people!" I think there is a lot truth in that statement. If we do not care about quality, timelines, good team culture, respectful communication (!!), clean code, if all this doesn’t matter to us, then I believe the probability is higher that we fail in research and analysis.

5. Be good with people
Because bioinformatician and computational biologist jobs typically involves to work in a (most wanted J cross-departmental!) team, therefore it's important that we're (more or less) good in dealing with other individuals. Everyone have their own strengths and weaknesses, just like us. It's important to treat all the research team mates with respect, regardless of their technical competence or contributions. Of course, sometimes people deserve a clear statement (!!!), but try to do these things one-on-one. Make sure nobody loses his face. Attend the meetings at the coffee bar; be good at table top soccer and go out once in a while to have a beer with your team. You know what I'm talking about.

At the end of a week I look back and I ask myself what I have produced. This could be paperwork, community days or (best!!) programming code. Always remember there is always a solution to a problem. Most of the times there are at least three solutions. So, don’t just blame, suggest a solution.

That's it. I am looking forward to your thoughts and comments!

minimap -t 24 assembly.fasta long_reads.fastq.gz | racon -t 24 long_reads.fastq.gz - assembly.fasta racon_assembly.fasta
nucmer -p nucmer assembly.fasta racon_assembly.fasta
show-snps -C -T -r nucmer.delta

This reports Racon's changes in a table. You can exclude indels with the -I option in show-snps. 

This process (Racon -> MUMmer -> SNP table) solves the problem I originally raised in this issue. So as far as I'm concerned, you can close this issue (or keep it open if you still want to implement some kind of variant table).

For knowledge about Linux and their importance amongst bioinformatician plesae read this article "An introduction to Linux for bioinformatics" by Paul Stothard.

Linux cheat sheet at http://bioinformaticsonline.com/file/view/87/linux-cheat-sheet

Please browse for futher useful linux pages on right hand side ...

We accomplish this by first calculating genotype likelihoods for every site. For a given site in a diploid genome, there are 10 possible genotypes (AA, AC, AG, AT, CC, CG, CT, GG, GT, TT). Referee takes as input the genotype likelihoods calculated for all 10 genotypes given the called reference base at each position.

Referee is a program to calculate a quality score for every position in a genome assembly. This allows for easy filtering of low quality sites for any downstream analysis.

https://github.com/gwct/referee

Address of the bookmark: https://gwct.github.io/referee/#

Screen is like a window manager for your console. It will allow you to keep multiple terminal sessions running and easily switch between them. It also protects you from disconnection, because the screen session doesn’t end when you get disconnected.

You’ll need to make sure that screen is installed on the server you are connecting to. If that server is Ubuntu or Debian, just use this command:

sudo apt-get install screen

Now you can start a new screen session by just typing screen at the command line. You’ll be shown some information about screen. Hit enter, and you’ll be at a normal prompt.

To disconnect (but leave the session running)

Hit Ctrl + A and then Ctrl + D in immediate succession. You will see the message [detached]

To reconnect to an already running session

screen -r

To reconnect to an existing session, or create a new one if none exists

screen -D -r

To create a new window inside of a running screen session

Hit Ctrl + A and then C in immediate succession. You will see a new prompt.

To switch from one screen window to another

Hit Ctrl + A and then Ctrl + A in immediate succession.

To list open screen windows

Hit Ctrl + A and then W in immediate succession

A swift, versatile phylogenomic and high-throughput sequencing simulator https://jackalope.lucasnell.com

Address of the bookmark: https://github.com/lucasnell/jackalope

'du' – Check the size of a directory

$ du
This command ( du) gives you a list of directories that exist in the current working directory along with their sizes in kilobytes (default). The last line of the output gives you the total size of the current directory including its subdirectories.

$ du /home/jin1
The above command would give you the directory size of the directory /home/david

$ du -h
The same “du”command with some flag gives you a better output than the default one. The option '-h' stands for human readable format. Therefore, in order to print the sizes of the files / directories in your desire notation use this time suffixed with a 'k' if its kilobytes and 'M' if its Megabytes and 'G' if its Gigabytes.

$ du -ah
If you are interested in checking everything present in a folder use above mentioned command. It gives us not only the directories but also all the files that are present in the current directory. The “-a” flag displays the filenames along with the directory names in the output.

$ du -c
This gives you a grand total as the last line of the output. So if your directory occupies 30MB the last 2 lines of the output would be 30M.

$ du -s
Use this command to displays a summary of the directory size. It is the simplest way to know the total size of the current directory.

$ du -S
This would display the size of the current directory excluding the size of the subdirectories that exist within that directory. So it basically shows you the total size of all the files that exist in the current directory.

$ du --exculde=mp3
Several times it required to exclude some directory in our size calculation. In such cases the above command would display the size of the current directory along with all its subdirectories, but it would exclude all the files having the given pattern present in their filenames.

'df' - finding the disk free space / disk usage

$ df
Hmmm … now “df” command is really useful, and I guess you are going to use it over time. Typing the above command, outputs a table consisting of 6 columns. All the columns are very easy to understand. Remember that the 'Size', 'Used' and 'Avail' columns use kilobytes as the unit. The 'Use%' column shows the usage as a percentage which is also very useful.

$ df -h
Displays the same output as the previous command but the '-h' indicates human readable format. Hence instead of kilobytes as the unit the output would have 'M' for Megabytes and 'G' for Gigabytes.

Example: Linux installed on /dev/hda1
$ df -h | grep /dev/hda1

All right, this is not the only option to check the sizes and free spaces but there are a few more options that can be used with 'du' and 'df' . I will discuss it later.

More at https://kevlar.readthedocs.io/en/latest/

https://www.cell.com/iscience/pdf/S2589-0042(19)30259-7.pdf

Address of the bookmark: https://github.com/kevlar-dev/kevlar