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.

Quickstart

http://nanopolish.readthedocs.io/en/latest/quickstart_consensus.html

Algorithms

http://simpsonlab.github.io/2017/06/30/nanopolish-v0.7.0/

Address of the bookmark: https://github.com/jts/nanopolish

It demonstrates how to use long PacBio sequencing reads to assemble a bacterial genome, and includes additional steps for circularising, trimming, finding plasmids, and correcting the assembly with short-read Illumina data.

 Please comment if you know any other long read addembly tutorial.

Address of the bookmark: http://sepsis-omics.github.io/tutorials/modules/cmdline_assembly_v2/

Address of the bookmark: https://github.com/fenderglass/Flye

https://academic.oup.com/nar/article/47/11/e63/5377471

Address of the bookmark: https://github.com/linzhi2013/MitoZ

More at https://software.broadinstitute.org/gatk/download/bundle

ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/VCF/GATK/.

ftp://ftp.broadinstitute.org/bundle/hg38/hg38bundle/

Address of the bookmark: https://console.cloud.google.com/storage/browser/genomics-public-data/resources/broad/hg38/v0?pli=1

.NET Bio

http://blogs.msdn.com/b/msr_er/archive/2011/10/18/microsoft-biology-foundation-evolves-into-new-toolkit-net-bio.aspx

A language-neutral bioinformatics toolkit built using the Microsoft 4.0 .NET Framework to help developers, researchers, and scientists.

AMPHORA ("AutoMated Phylogenomic infeRence Application")

http://wolbachia.biology.virginia.edu/WuLab/Software.html

Metagenomics analysis software

Anduril

http://www.anduril.org/anduril/site/

Component-based workflow framework for data analysis

Armadillo workflow platform

Tool for designing and executing phylogenetic workflows

AutoDock

http://autodock.scripps.edu/

suite of automated docking tools

Biochemical Algorithms Library (BALL)

http://www.ball-project.org/

C++ library and framework for molecular modeling and visualization designed for rapid prototyping

Bio4j

http://bio4j.com/

Bio4j is a bioinformatics platform and graph based database built around most data available in UniProt KB(Swiss-Prot + TrEMBL), Gene Ontology (GO), UniRef (50,90,100), RefSeq, NCBI taxonomy, and Expasy Enzyme DB

Bioclipse

www.bioclipse.net

Visual platform for chemo- and bioinformatics based on the Eclipse Rich Client Platform (RCP).

Bioconductor

http://www.bioconductor.org/

R (programming language) language toolkit

Bioinformatics Learning Tutorial (BLT)

http://sourceforge.net/projects/biotutorial/

Educational interactive tutorials and 3D animations for Replication, Transcription, and Translation

BioHaskell

http://biohaskell.org/

Haskell (programming language)

BioJava

http://biojava.org/wiki/Main_Page

Java (programming language)

BioMOBY

http://biomoby.org/

registry of web services

BioPerl

http://www.bioperl.org/wiki/Main_Page

Perl language toolkit

BioPHP

http://www.biophp.org/

PHP language toolkit

Biopython

http://biopython.org/wiki/Main_Page

Python language toolkit

BioRails

https://github.com/biorails

a data management system designed to support researchers in drug discovery

BioRuby

http://bioruby.org/

Ruby language toolkit

BioSmalltalk

https://code.google.com/p/biosmalltalk/

Smalltalk language toolkit

BioUno

http://www.biouno.org/

BioUno is a project that applies Continuous Integration tools and techniques in Bioinformatics. It uses Jenkins and its plug-in API to create biology workflows and manage computer clusters.

caCORE

ontologic representation environment

caArray

https://cabig-stage.nci.nih.gov/community/tools/caArray

ontologic representation environment

EMBOSS

http://emboss.sourceforge.net/

Suite of packages for sequencing, searching, etc.

Gaggle

https://www.gaggle.net/

A framework for interoperability between systems biology software

Galaxy

http://galaxyproject.org/

Scientific workflow and data integration system

GenePattern

http://www.broadinstitute.org/cancer/software/genepattern/

Scientific workflow system that provides access to more than 150 genomic analysis tools

GeWorkbench

http://wiki.c2b2.columbia.edu/workbench/index.php/Home

Genomic data integration platform

GMOD

http://www.gmod.org/wiki/Main_Page

Toolkit for addressing many common challenges at biological databases.

GeneProf

http://www.geneprof.org/GeneProf/

A web-based, bioinformatics software suite for the analysis of functional genomics experiments, e.g. RNA-seq or ChIP-seq.

GeneTalk

http://www.gene-talk.de/

Tool for filtering sequence variants in VCF files. Network for scientists and clinicians for expertise and knowledge exchange. Database of annotations aboute sequence variants with clinically relevant information.

GenGIS

http://kiwi.cs.dal.ca/GenGIS/Main_Page

Application that allows users to combine digital map data with information about biological sequences collected from the environment.

GenomeSpace

http://www.genomespace.org/

Centralized web application that provides data format transformations and facilitates connections with other bioinformatics tools

GENtle

http://directory.fsf.org/wiki/GENtle

An equivalent to the proprietary Vector NTI, a tool to analyze and edit DNA sequence files

Integrated Genome Browser

http://bioviz.org/igb/

Java-based desktop genome browser

Integrative Genomics Viewer (IGV)

http://www.broadinstitute.org/igv/

High-performance desktop tool for interactive visual exploration of diverse genomic data

IntAct

http://www.ebi.ac.uk/intact/

molecular interaction database

InterMine

http://intermine.github.io/intermine.org/

Extensive data warehouse system for the analysis and integration of biological datasets

Java Treeview

http://jtreeview.sourceforge.net/

microarray data viewer

LabKey Server

http://labkey.com/

platform for integrating, analyzing and sharing data

OpenClinica

https://www.openclinica.com/

software for capturing and managing data in clinical trials

PromKappa

http://xbioinformatics.wordpress.com/tag/promkappa/

PromKappa (Promoter analysis by Kappa) software program used for promoter pattern generation and promoter analysis.

MeV: Multi-Experiment Viewer

http://www.tm4.org/mev.html

a desktop application for the analysis, visualization and data-mining of large-scale genomic data

PathVisio

http://www.pathvisio.org/

a desktop software for drawing, analysis and visualization of biological pathways

REDCRAFT

software for determining tertiary protein structure given assigned Residual Dipolar Coupling data

SAM Tools

Data format (SAM) and accompanying tool suite, for storing large nucleotide sequence alignments

Staden Package

Sequence assembly, editing and analysis, primarily consisting of gap4, gap5 and spin.

STAMP

Software package for analyzing metagenomic profiles that promotes ‘best practices’ in choosing appropriate statistical techniques and reporting results.

supraHex

An open-source R/Bioconductor package for omics data analysis using a supra-hexagonal map

Taverna workbench

Tool for designing and executing workflows

TGAC Browser

Genome Browser, visualisation solutions for big data in the genomic era

T-REX WebServer

Bioinformatics and phylogenetics webserver (NJ, PhyML, RAxML, MAFFT, MUSCLE, Newick viewer, Horizontal gene transfer detection, Reticulograms, Substitution models)

UGENE

integrated bioinformatics tools

Visomics

bioinformatics tools for omics data

Genome Analysis Toolkit 1.0 (GATK 1.0)

a software package to analyse next-generation resequencing data

Spines

http://www.broadinstitute.org/science/programs/genome-biology/spines

Address of the bookmark: http://www.broadinstitute.org/science/programs/genome-biology/spines

Arlequin: http://cmpg.unibe.ch/software/arlequin3/

BEAGLE: http://faculty.washington.edu/browning/beagle/beagle.html

fastPHASE: http://stephenslab.uchicago.edu/software.html

GENEHUNTER: http://linkage.rockefeller.edu/soft/gh/

The Genome Analysis Toolkit:

http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit

IMPUTE2: https://mathgen.stats.ox.ac.uk/impute/impute_v2.html

MACH: http://www.sph.umich.edu/csg/abecasis/MACH/

MERLIN: http://www.sph.umich.edu/csg/abecasis/Merlin/

PHASE: http://stephenslab.uchicago.edu/software.html

PL-EM: http://www.people.fas.harvard.edu/~junliu/plem/

“Read-backed phasing” algorithm: http://www.broadinstitute.org/gsa/wiki/index.php/Read-backed_phasing_algorithm

SHAPE-IT: http://www.griv.org/shapeit/

The MEME Suite allows the biologist to discover novel motifs in collections of unaligned nucleotide or protein sequences, and to perform a wide variety of other motif-based analyses.

The MEME Suite supports motif-based analysis of DNA, RNA and protein sequences. It provides motif discovery algorithms using both probabilistic (MEME) and discrete models (MEME), which have complementary strengths. It also allows discovery of motifs with arbitrary insertions and deletions (GLAM2). In addition to motif discovery, the MEME Suite provides tools for scanning sequences for matches to motifs (FIMO, MAST and GLAM2Scan), scanning for clusters of motifs (MCAST), comparing motifs to known motifs (Tomtom), finding preferred spacings between motifs (SpaMo), predicting the biological roles of motifs (GOMo), measuring the positional enrichment of sequences for known motifs (CentriMo), and analyzing ChIP-seq and other large datasets (MEME-ChIP).

The MEME Suite is comprised of a collection of tools that work together, as shown below. Not all the tools are available as webservices, so to get the full power of the MEME Suite you will need to download and install a local copy of the software. To see what has changed recently you can peruse the release notes.

http://meme-suite.org/

Address of the bookmark: http://meme-suite.org/