Hands-on introduction to NGS variant analysis-2020
- 1 Aims of the NGS DNA variant analysis 2-days session
- 2 Summary
- 3 Prerequisites
- 4 Hands-On Exercises
- 5 Answers to your 2017 requests
- 6 PDF version of the former training
- 7 Find more tools and answers
- 8 Extra readings
- 9 GATK performance in numbers
- 10 Conclusion & Contact
Aims of the NGS DNA variant analysis 2-days session
Using a full publicly available chromosome read-set from one of the 1000 genomes samples:
- Use the graphical environment provided by the BITS GenePattern server to perform all steps of a classical NGS variant workflow and feel the complexity of the task.
- Perform a simplified analysis workflow including read mapping, variant calling against the current human reference genome
- Annotate the obtained variant calls and compare them to the public variant file for that genome.
- implicitly: get the motivation to go to the next level and learn command line and R
Scheme of today's workflow
More BITS Training Info
- On the VIB website: http://www.vib.be/en/training/research-training/courses/Pages/Hands-On-introduction-to-NGS-variant-analysis.aspx
- On the BITS website: https://www.bits.vib.be/index.php/training/201-ngs-variant-analysis
This training gives an introduction to the use of popular NGS analysis software packages through the GenePattern graphical interface. It reviews several exchangeable tools and provides hints to evaluate quality and content of Genome-Seq data. Much more can be (and should be) done when working at command-line and GenePattern will not replace advanced use of the terminal. However, this simplified workflow will allow unexperienced scientists to discover the practices involved with variant analysis. A recent review by Geraldine Van der Auwera et al develops on many aspects of this .
The sequencing data used in this session was obtained from high coverage depth Illumina HiSeq sequencing of gDNA extracted from EBV-transformed B-lymphocytes of a healthy CEPH/UTAH Mormon mother (NA12878). More information about that sample is available from the Coriell repository from which all 1000g gDNA can be obtained .
The paired end reads used in this training were extracted from the Genome In A Bottle (GIAB) novoalign BAM mappings of Illumina HiSeq 300x reads for NA12878 available on the ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/ server . This pre-processing step is not covered in the training. A lot of analysis results are available on the dedicated GIAB page 
We use this year the state of the art Genome Analysis Toolkit ('GATK') for variant analysis and we strongly advise you to consider it in your work. GATK was compared to varscan used in former training sessions and is clearly superior in sensitivity and specificity . GATK is today the standard workflow for human genome analyses and is referred in most publications. We do not apply here all the available tricks GATK allows but rather present you a streamline pipeline with a single genome that will be a good foundation for most applications. Please read the GATK pages for more information.
The official GATK best practice inspiring this training is as follows:
Translated into separate exercises from today's training (red numbers next to boxes)
Disclaimer: This training does not cover all currently available methods. It does not aim at bringing you to a professional NGS analyst level but provides enough information to allow a motivated biologist understanding what DNA sequencing practically is, and when necessary to communicate knowingly with NGS experts for more in-depth needs.
Skills required to follow this training
- Linux command line basic skills are required to review some of the long text results under terminal (GenePattern is not handy in reviewing data, it is mainly a job submission and management platform)
- basic knowledge of human genome structure and nomenclature is necessary to understand the training tasks and the results
- basic knowledge of Illumina NGS read structure is also required for the same reason
Software and Arguments used during this training
- All programs used in this training session were installed on the BITS GenePattern server and specific modules where created for you by Guy Bottu (BITS). These modules are therefore absent from other GenePattern servers like those accessible at the Broad. If you plan to build your own GenePattern Server (GP home page), you may ask Guy copies of these modules contact us.
- The GATK4 Best Practices are a series of modules developed at the Broad to analyse sequencing data and are subject to regular changes.
At the time of preparing this training we obtained information from this link
The information used to build the Genepattern modules for this training were obtained from the link shown above and pointing to the Best Practices Wiki pages and the corresponding optional arguments extracted from the WDL scripts posted there.
REM: At the end of each page, a link to data available on our file server is added and allows downloading some of the data for local use and self-training. Other files, required for the training are present on the server and will be accessed directly from within Genepattern.
- NGS-Var2020 Startup GenePattern: Locate data and tools in your training account
- NGS-Var2020 Exercise.1: QC paired end reads using fastQC
- NGS-Var2020 Exercise.2: Map to the human reference genome GRCh38 using the Burrow Wheeler Aligner (BWA) and recalibrate the mappings with Picard and GATK4
- NGS-Var2020 Exercise.3: Perform QC on the mapping data with Picard, and Qualimap
- NGS-Var2020 Exercise.4: Call variants and produce genotypes with customised Genepattern modules based on the GATK best practices
- NGS-Var2020 Exercise.5: Intersect raw VCF files using VCF_intersect
- NGS-Var2020 Exercise.6: Annotate VCF variant lists with SNPSift and filter candidates with SnpEff
- NGS-Var2020 Exercise.7: Review read alignments, gene annotations, and variants calls in IGV
Answers to your 2017 requests
Some of you asked about the possibility to call variants from structured experiments like family trios or tumor-normal pairs. We found trio data for one of the public 1000 genome trio (CEPH CEU) as well as a tumor-normal pancreatic cancer dataset (WES) with which we prepared two walk-through tutorial for command-line analysis using Varscan2. Both documents have been linked below.
- call variants from a family trio (NA12878) and identify inherited and potential non-inherited variants (de-novo) using Varscan 2 (link)
- call variants from a pair of samples (tumor and normal tissues of the same patient) and identify somatic variants and CNA (copy number abnormalities) in the tumor sample (link)
PDF version of the former training
- A partial PDF export of these pages can be downloaded here
- A LatEx tutorial to perform a similar analysis at command-line can be found here
Find more tools and answers
There are many tools out there, finding them is often the easiest part. You are welcome to try as many as you wish and improve results obtained with our selected toolbox. When seeking advice, please consider using:
- SeqAnswers for all what relates to NGS
- BioStar for questions about biocomputing and scripting for biologists
- stackoverflow for questions related to coding.
- The EBI online training propose many very nice training sessions with slides and exercises.
- A number of reference files are used to run GATK. They are present on our Genepattern server but if you want to run GATK on your own machine you will need to get these files. Among others, the GATK Bundle can be accessed here
- Another recent BMC Bioinformatics paper  reviews ways to accelerate your pipeline.
Other packages have been compared including mappers and callers, here is a starter for more readings, you will find more on Google.
- A Comparison of Variant Calling Pipelines Using Genome in a Bottle as a Reference 
- Performance Assessment of Variant Calling Pipelines using Human Whole Exome Sequencing and Simulated data 
- VCF-Miner: GUI-based application for mining variants and annotations stored in VCF files 
- Evaluating Variant Calling Tools for Non-Matched Next-Generation Sequencing Data 
- appreci8: a pipeline for precise variant calling integrating 8 tools 
What about longer variants (structural!)?
- Accurate detection of complex structural variations using single-molecule sequencing 
What about CNV and RNASeq variants?
- GATK4 is developing new BestPractices including
GATK performance in numbers
A recent nature biotechnology paper by Poplin et al  compared GATK to other callers. Their results concerning GATK (version 3.8) are pictured below from the first two tables.
Conclusion & Contact
Your feedback to this introductory NGS variant analysis using GenePattern session is very important to us and will be used to improve this content for later sessions. If you need more training of this kind, please contact us and we will organise additional hands-on based on your requests. More advance sessions will depend on the availability of expert users within VIB that will accept to prepare specified material.
- http://seqanswers.com SeqAnswers
- http://www.biostars.org BioStar
- http://stackoverflow.com stackoverflow
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