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.

Address of the bookmark: https://peerj.com/preprints/453.pdf

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).

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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/#

Apply now Job no: 494553
Work type: Continuing full time
Vacancy type: External Vacancy, Internal Vacancy
Categories: Academic - Teaching and Research

The Faculty of Science is launching a new and innovative branch of biological science at Macquarie University – Synthetic Biology. Synthetic biology combines engineering principles with molecular biological approaches to design and construct biological devices and systems. Recent highlights in this field include the design and synthesis of a functional bacterial genome and a yeast chromosome, and generation of synthetic bacterial cells. The rational synthesis of "designer" organisms yield important insights into how organisms work and has the potential to revolutionise biotechnological applications in areas such as bioenergy and biomanufacturing.

Find more at http://jobs.mq.edu.au/cw/en/job/494553/lecturersenior-lecturer-level-bc-in-synthetic-biology-research-fellow-level-b-in-synthetic-biology-lecturersenior-lecturer-level-bc-in-bioinformatics

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

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

Applicants are invited to send their resume and a recommendation letter to Prof. Patrick Meyer (more details at www.biosys.ulg.ac.be )

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

This job will allow the intern to become more familiar with new biological and bioinformatics tools like next generation sequencing, RNA-Seq data analysis and comparative genomics.

To apply for this position, send the following documents (in PDF format) to Dr Pierre-Yves Dupont (email p.y.dupont@massey.ac.nz):

1. A short cover letter.
2. A curriculum vitae, with transcript details.
3. The names and contact details of two referees willing to provide a confidential letter of recommendation upon request.

Informal enquiries are welcome. Formal applications are due by Sunday 2nd December 2012.
Requirements:

This position requires a good understanding of genetic problems, a good command of at least one scripting language (Perl, Python...), a basic knowledge of MySQL or any relational database management system. Knowledge in biological programming libraries (BioPython, BioPerl, BioRuby...), Java, C++ or any compiled language is an asset but not required. Undergraduate or Master degree is required.
Contact Information:

Dr. Pierre-Yves Dupont
Institute of Molecular BioSciences
Massey University
Private Bag 11 222
Palmerston North 4442
NEW ZEALAND

http://massey.genomicus.com/
p.y.dupont@massey.ac.nz

Information about the Institute of Molecular BioSciences (http://imbs.massey.ac.nz/) and the Computational Biology Research Group (http://massey.genomicus.com/) is available online. For more information about the position, you can contact Dr Pierre-Yves Dupont (email p.y.dupont@massey.ac.nz).