Project Scientist-I
Experimental / Computation analysis experience in highthroughput genomics/ clinical application.

Project Manager
Experience in handling large biological projects involving high-throughput genomics/ clinical application.

Scientific Administrative Assistant
Lab Work.

More at https://vinodscaria.genomes.in/positionsopen

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.

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).

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/#

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

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

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

What Are Kallisto and Salmon?

At their core, both Kallisto and Salmon are tools for quantifying transcript abundance from RNA-Seq reads. They bypass traditional alignment-based methods, replacing them with pseudoalignment or quasi-mapping, which drastically speeds up the process.

• Kallisto was developed by Lior Pachter’s lab and introduced the concept of pseudoalignment using a de Bruijn graph.
• Salmon, developed by Rob Patro’s group, builds on this idea with quasi-mapping and offers additional features like advanced bias correction.

Head-to-Head Comparison

1. Algorithm

• Kallisto uses pseudoalignment, focusing on matching k-mers from reads to a transcriptome index.
• Salmon uses quasi-mapping, which adds more flexibility and can also work with aligned reads (BAM files).

2. Input and Flexibility

• Kallisto works with raw FASTQ reads and requires a custom transcriptome index.
• Salmon accepts FASTQ or pre-aligned BAM files, giving you more workflow options.

3. Bias Correction

One of Salmon’s major advantages is its sophisticated bias correction system. It corrects for:

• Sequence-specific bias
• Positional bias
• GC-content bias

Kallisto offers basic sequence bias correction but lacks the comprehensive models found in Salmon.

4. Speed and Resources

• Kallisto is blazing fast and slightly more memory-efficient.
• Salmon is still very fast, but the added features can come at a small computational cost.

5. Output and Downstream Analysis

• Both tools provide transcript-level quantifications and support bootstrapping for variance estimation.
• Salmon can also summarize counts at the gene level if provided with a mapping file (--geneMap).
• Kallisto integrates seamlessly with Sleuth for differential expression analysis.
• Salmon works well with tximport, DESeq2, edgeR, and other Bioconductor tools.

Choosing the Right Tool

Example Command Lines

Kallisto (paired-end):

kallisto quant -i transcriptome.idx -o output -b 100 sample_R1.fastq sample_R2.fastq

Salmon (paired-end, bias correction):

salmon quant -i salmon_index -l A -1 sample_R1.fastq -2 sample_R2.fastq \
  -p 8 --validateMappings --seqBias --gcBias -o output

Conclusion

Both Kallisto and Salmon are exceptional tools that have transformed RNA-Seq analysis. Your choice largely depends on your priorities—whether it's speed, accuracy, flexibility, or compatibility with downstream tools.

For many users, Salmon offers a more complete and flexible solution, especially when bias correction and gene-level outputs are essential. However, Kallisto remains a favorite for quick, accurate quantification, especially when paired with the Sleuth pipeline.

Use the Terminal or an Anaconda Prompt for the following steps.

1. To create an environment:

   conda create --name myenv
   

   NOTE: Replace myenv with the environment name.

2. When conda asks you to proceed, type y:

   proceed ([y]/n)?
   

This creates the myenv environment in /envs/. This environment uses the same version of Python that you are currently using, because you did not specify a version.

To create an environment with a specific version of Python:

conda create -n myenv python=3.4

To create an environment with a specific package:

conda create -n myenv scipy

OR:

conda create -n myenv python
conda install -n myenv scipy

To create an environment with a specific version of a package:

conda create -n myenv scipy=0.15.0

OR:

conda create -n myenv python
conda install -n myenv scipy=0.15.0

To create an environment with a specific version of Python and multiple packages:

conda create -n myenv python=3.4 scipy=0.15.0 astroid babel

TIP: Install all the programs that you want in this environment at the same time. Installing 1 program at a time can lead to dependency conflicts.

To automatically install pip or another program every time a new environment is created, add the default programs to the create_default_packages section of your .condarc configuration file. The default packages are installed every time you create a new environment. If you do not want the default packages installed in a particular environment, use the --no-default-packages flag:

conda create --no-default-packages -n myenv python

TIP: You can add much more to the conda create command. For details, run conda create --help.

➜ redundans git:(master) ✗ conda create --name py27 python=2.7
Solving environment: done

==> WARNING: A newer version of conda exists. <==
current version: 4.5.0
latest version: 4.5.2

Please update conda by running

$ conda update -n base conda

## Package Plan ##

environment location: /home/urbe/anaconda3/envs/py27

added / updated specs:
- python=2.7

The following packages will be downloaded:

package | build
---------------------------|-----------------
wheel-0.31.0 | py27_0 61 KB
python-2.7.15 | h1571d57_0 12.1 MB
certifi-2018.4.16 | py27_0 142 KB
sqlite-3.23.1 | he433501_0 1.5 MB
setuptools-39.1.0 | py27_0 582 KB
openssl-1.0.2o | h20670df_0 3.4 MB
pip-10.0.1 | py27_0 1.7 MB
ca-certificates-2018.03.07 | 0 124 KB
------------------------------------------------------------
Total: 19.6 MB

The following NEW packages will be INSTALLED:

ca-certificates: 2018.03.07-0
certifi: 2018.4.16-py27_0
libedit: 3.1-heed3624_0
libffi: 3.2.1-hd88cf55_4
libgcc-ng: 7.2.0-hdf63c60_3
libstdcxx-ng: 7.2.0-hdf63c60_3
ncurses: 6.0-h9df7e31_2
openssl: 1.0.2o-h20670df_0
pip: 10.0.1-py27_0
python: 2.7.15-h1571d57_0
readline: 7.0-ha6073c6_4
setuptools: 39.1.0-py27_0
sqlite: 3.23.1-he433501_0
tk: 8.6.7-hc745277_3
wheel: 0.31.0-py27_0
zlib: 1.2.11-ha838bed_2

Proceed ([y]/n)? y

Downloading and Extracting Packages
wheel 0.31.0: #################################################################################################################################################################################################### | 100%
python 2.7.15: ################################################################################################################################################################################################### | 100%
certifi 2018.4.16: ############################################################################################################################################################################################### | 100%
sqlite 3.23.1: ################################################################################################################################################################################################### | 100%
setuptools 39.1.0: ############################################################################################################################################################################################### | 100%
openssl 1.0.2o: ################################################################################################################################################################################################## | 100%
pip 10.0.1: ###################################################################################################################################################################################################### | 100%
ca-certificates 2018.03.07: ###################################################################################################################################################################################### | 100%
Preparing transaction: done
Verifying transaction: done
Executing transaction: done
#
# To activate this environment, use:
# > source activate py27
#
# To deactivate an active environment, use:
# > source deactivate
#

➜ redundans git:(master) ✗ source activate py27

Is this available, or still under trial mode? 

https://www.nanoporetech.com/

https://www.nanoporetech.com/technology/the-minion-device-a-miniaturised-sensing-system/the-minion-device-a-miniaturised-sensing-system