Data analysis and interpretation for clinical genomics

Overview

question Questions
  • What are the specific challenges for the interpretation of sequencing data in the clinical setting?

  • How can you annotate variants in a clinically-oriented perspective?

objectives Objectives
  • Perform in-depth quality control of sequencing data at multiple levels (fastq, bam, vcf)

  • Call, classify and annotate variants with information extracted from public databases for clinical interpretation

  • Analyze CNV and Regions of Homozygosity (ROH)

time Time estimation: 6 hours

last_modification Last modification: Jan 24, 2020

Introduction

In years 2018-2019, on behalf of the Italian Society of Human Genetics (SIGU) an itinerant Galaxy-based “hands-on-computer” training activity entitled “Data analysis and interpretation for clinical genomics” was held four times on invitation from different Italian institutions (Università Cattolica del Sacro Cuore in Rome, University of Genova, SIGU 2018 annual scientific meeting in Catania, University of Bari) and was offered to about 30 participants each time among clinical doctors, biologists, laboratory technicians and bioinformaticians. Topics covered by the course were NGS data quality check, detection of variants, copy number alterations and runs of homozygosity, annotation and filtering and clinical interpretation of sequencing results.

Realizing the constant need for training on NGS analysis and interpretation of sequencing data in the clinical setting, we designed an on-line Galaxy-based training resource articulated in presentations and practical assignments by which students will learn how to approach NGS data quality at the level of fastq, bam and VCF files and clinically-oriented examination of variants emerging from sequencing experiments.

This training course is not to be intended as a tutorial on NGS pipelines and variant calling. This on-line training activity is indeed focused on data analysis for clinical interpretation. If you are looking for training on variant calling, visit this Galaxy tutorial on Exome sequencing data analysis for diagnosing a genetic disease.

comment SIGU

The Italian Society of Human Genetics (SIGU) was established on November 14, 1997, when the pre-existing Italian Association of Medical Genetics and the Italian Association of Medical Cytogenetics joined. SIGU is one of the 27 member societies of FEGS (Federation of European Genetic Societies). Animated by a predominant scientific spirit, SIGU wants to be reference for all health-care issues involving human genetics in all its applications. Its specific missions are to develop quality criteria for medical genetic laboratories, to promote writing of guidelines in the field of human genetics and public awareness of the role and limitations of genetic diagnostic techniques. SIGU coordinates activities of several working groups: Clinical Genetics, Cytogenetics, Prenatal Diagnosis, Neurogenetics, Fingerprinting, Oncological Genetics, Immunogenetics, Genetic Counseling, Quality Control, Medical Genetics Services, Bioethics. More than 1000 medical geneticists and biologists are active members of the society.

Agenda

In this tutorial, we will cover:

  1. Requirements
  2. Datasets
    1. Get data
  3. Next Generation Sequencing
    1. NGS in the clinic
    2. Basics of NGS bioinformatic analysis
    3. Alignment
    4. Post-alignment BAM processing
    5. Variant calling
  4. Quality control
    1. Quality control of FASTQ files
    2. Quality control of BAM files
  5. Variant calling and classification
  6. Variant annotation
    1. Gene model
    2. Sequence variant nomenclature
    3. Variant class
    4. Population sequencing db
    5. Variant-disease/gene-disease db
    6. Annotation Software and tools
    7. Annotation and filtering with wANNOVAR
    8. Clinical databases for further manual variant annotation
  7. Variant prioritization
    1. Variant impact
    2. Variant frequency
    3. Variant effect prediction Tools
    4. ACMG/AMP 2015 guidelines
    5. Prioritization
  8. CNV detection from targeted sequencing data
    1. Computational approaches
    2. RC method and data normalization
    3. CNV detection accuracy
    4. Tools for CNV detection from gene panels or exome data
  9. Regions of Homozygosity
    1. Homozygosity mapping
    2. Computational approaches

Requirements

This tutorial is based on the Galaxy platform, therefore a basic knowledge of Galaxy is required to get most out of the course. In particular, we’ll use European Galaxy server running at https://usegalaxy.eu.

Registration is free, and you get access to 250GB of disk space for your analysis.

  1. Open your browser. We recommend Chrome or Firefox (please don’t use Internet Explorer or Safari).
  2. Go to https://usegalaxy.eu
    • If you have previously registered on this server just log in:
      • On the top menu select: User -> Login
      • Enter your user/password
      • Click Submit
    • If you haven’t registered on this server, you’ll need to do now.
      • On the top menu select: User -> Register
      • Enter your email, choose a password, repeat it and add a one word name (lower case)
      • Click Submit

To familiarize with the Galaxy interface (e.g. working with histories, importing dataset), we suggest to follow the Galaxy 101 tutorial.

Datasets

Input datasets used in this course are available:

Get data

hands_on Hands-on: Data upload

  1. Create a new history for this tutorial and give it a meaningful name (e.g. Clinical genomics)

    tip Tip: Creating a new history

    Click the new-history icon at the top of the history panel

    If the new-history is missing:

    1. Click on the galaxy-gear icon (History options) on the top of the history panel
    2. Select the option Create New from the menu

    tip Tip: Renaming a history

    1. Click on Unnamed history (or the current name of the history) (Click to rename history) at the top of your history panel
    2. Type the new name
    3. Press Enter
  2. Import files from Zenodo or Shared Data Library:

    https://zenodo.org/record/3531578/files/CNV_case.bam
    https://zenodo.org/record/3531578/files/CNV_control.bam
    https://zenodo.org/record/3531578/files/CNV_TruSeq_Chr2.bed
    https://zenodo.org/record/3531578/files/HighQuality_Reads.fastq.gz
    https://zenodo.org/record/3531578/files/LowQuality_Reads.fastq.gz
    https://zenodo.org/record/3531578/files/Panel_alignment.bam
    https://zenodo.org/record/3531578/files/Panel_target_regions.bed
    https://zenodo.org/record/3531578/files/Sample1.all_exons.hg19.vcf
    
    • Copy the link location
    • Open the Galaxy Upload Manager (galaxy-upload on the top-right of the tool panel)

    • Select Paste/Fetch Data
    • Paste the link into the text field

    • Press Start

    • Close the window

    By default, Galaxy uses the URL as the name, so rename the files with a more useful name.

    The same files may be available on the Galaxy server through a Shared Data Libraries in Galaxy courses/Sigu. You may prefer to import the data directly from there.

    tip Tip: Importing data from a data library

    As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:

    • Go into Shared data (top panel) then Data libraries

    • Find the correct folder (ask your instructor)

    • Select the desired files
    • Click on the To History button near the top and select as Datasets from the dropdown menu
    • In the pop-up window, select the history you want to import the files to (or create a new one)
    • Click on Import

    comment Note

    All the files are based on hg19 reference genome which is available with pre-built indexes for widely used tools such as bwa-mem and samtools by selecting hg19 version as an option under “(Using) reference genome”).

  3. In case you import datasets from Zenodo, check that all datasets in your history have their datatypes assigned correctly, and fix it when necessary. For example, to assign BED datatype do the following:

    tip Tip: Changing the datatype

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, click on the galaxy-chart-select-data Datatypes tab on the top
    • Select bed
    • Click the Change datatype button
  4. Rename the datasets

    For datasets uploaded via a link, Galaxy will use the link as the dataset name. In this case you may rename datasets.

    tip Tip: Renaming a dataset

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, change the Name field
    • Click the Save button

Next Generation Sequencing

Next (or Second) Generation Sequencing (NGS/SGS) is an umbrella-term covering a number of approaches to DNA sequencing that have been developed after the first, widespread and for long time most commonly used Sanger sequencing.

NGS is also known as Massive Parallel Sequencing (MPS), a term that makes explicit the paradigm shared by all these technologies, that is to sequence in parallel a massive library of spatially separated and clonally amplified DNA templates.

For a comprehensive review of the different NGS technologies see Goodwin et al., 2016, which also includes an introduction to the third generation methods allowing sequencing of long single-molecule reads.

NGS in the clinic

In the span of less than a decade, NGS approaches have pervaded clinical laboratories revolutionizing genomic diagnostics and increasing yield and timeliness of genetic tests.

In the context of disorders with a recognized strong genetic contribution such as neurogenetic diseases, NGS has been firmly established as the strategy of choice to rapidly and efficiently diagnose diseases with a Mendelian basis. A general diagnostic workflow for these disorders currently embraces different NGS-based diagnostic options as illustrated in Figure 1.


ngs_neuro_diagnostics

Figure 1. General workflow for genetic diagnosis of neurological diseases. (*If considering high-yield single-gene testing of more than 1–3 genes by another sequencing method, note that next-generation sequencing is often most cost-effective. †Genetic counselling is required before and after all genetic testing; other considerations include the potential for secondary findings in genomic testing, testing parents if inheritance is sporadic or recessive, and specialty referral.) From Rexach et al., 2019


Currently, most common NGS strategies in clinical laboratories are the so-called targeted sequencing methods that, as opposed to genome sequencing covering the whole genomic sequence, focus on a pre-defined set of regions of interest (the targets). The targets can be selected by hybrid capture or amplicon sequencing, and the target-enriched libraries is then sequenced. The most popular target designs are:

  • gene panels where the coding exons of only a clinically-relevant group of genes are targeted
  • exome sequencing where virtually all the protein-coding exons in a genome are simultaneously sequenced

Basics of NGS bioinformatic analysis

Apart from the different width of the target space in exome and gene panels, these two approaches usually share the same experimental procedure for NGS library preparation. After clonal amplification, the fragmented and adapter-ligated DNA templates are sequenced from both ends (paired-end sequencing) of the insert to produce short reads in opposite (forward and reverse) orientation.

Bioinformatic analysis of NGS data usually follows a general three-step workflow to variant detection. Each of these three steps is marked by its “milestone” file type containing sequence data in different formats and metadata describing sequence-related information collected during the analysis step that leads to generation of that file.

NGS workflow step File content File format File Size (individual exome)
Sample to reads Unaligned reads and qualities fastQ gigabytes
Reads to alignments Aligned reads and metadata BAM gigabytes
Alignments to variants Genotyped variants and metadata VCF megabytes

Here are the different formats explained:

  • fastQ (sequence with quality): the de facto standard for storing the output of high-throughput sequencing machines
    • Usually not inspected during data analysis
  • BAM (binary sequence alignment/map): the most widely used TAB-delimited file format to store alignments onto a reference sequence
    • Aligned reads
  • VCF (variant call format): the standard TAB-delimited format for genotype information associated with each reported genomic position where a variant call has been recorded

Another useful file format is BED, to list genomic regions of interest such as the exome or panel targets.

The steps of the reads-to-variants workflow can be connected through a bioinformatic pipeline (Leipzig et al., 2017), consisting in read alignments, post-alignment BAM processing and variant calling.

Alignment

As generated by the sequencing machines, paired-end reads are written to two fastQ files in which forward and reverse reads are stored separately together with their qualities. FastQ files are taken as input files by tools (the aligners) that align the reads onto a reference genome. One of the most used aligners is BWA among the many that have been developed (Figure 2).


aligners_timeline

Figure 2. Aligners timeline 2001-2012 (from Fonseca et al., 2012)


During the bioinformatic process, paired-end reads from the two separate fastQ files are re-connected in the alignment, where it is expected that they will:

  • map to their correct location in the genome
  • be as distant as the insert size of the fragment they come from
  • be in opposite orientations a combination which we refer to as proper pairing. All these data about paired-end reads mapping are stored in the BAM file and can be used to various purposes, from alignment quality assessment to structural variant detection.

In Figure 3, the Integrative Genomic Viewer (IGV) screenshot of an exome alignment data over two adjacent ASXL1 exons is shown. Pink and violet bars are forward and reverse reads, respectively. The thin grey link between them indicates that they are paired-end reads. The stack of reads is concentrated where exons are as expected in an exome, and the number of read bases covering a given genomic location e (depicted as a hill-shaped profile at the top of the figure) defines the depth of coverage (DoC) over that location:

DoCe=number of read bases over e/genomic length of e


coverage Figure 3. Exome data visualization by IGV


Post-alignment BAM processing

Regarding post-alignment pipelines, the most famous for germline SNP and InDel calling is probably that developed as part of the GATK toolkit (Figure 4).


gatk_germline_pipeline

Figure 4. After-alignment pipeline for germline SNP and InDel variant calling according to GATK Best Practices


According to GATK best practices, in order to be ready for variant calling the BAM file should undergo the following processing:

  • marking duplicate reads to flag (or discard) reads that are mere optical or PCR-mediated duplications of the actual reads
  • recalibrating base quality scores to correct known biases of the native base-related qualities While GATK BAM processing is beyond doubt important to improve data quality, it has to be noticed that it is not needed to obtain variant calls and that non GATK-based pipelines may not use it or may use different quality reparametrization schemes. Duplicate flagging or removal is not recommended in amplicon sequencing experiments.

Variant calling

The process of variant detection and genotyping is performed by variant callers. These tools use probabilistic approaches to collect evidence that non-reference read bases accumulating over a given locus support the presence of a variant, usually differing in algorithms, filtering strategies, recommendations (Sandmann et al., 2017). To be confident that a variant is a true event, its supporting evidence should be significantly stronger than chance; e.g. the C>T on the left of the screenshot in Figure 5 is supported by all its position-overlapping reads, claiming for a variant. In contrast, the C>A change on the right of the screenshot is seen only once over many reads, challenging its interpretation as a real variant. In fact, DNA variants that occur in germ cells (i.e., germline/constitutional variants that can be passed on to offspring) are either diploid/biallelic, so expected alternative allele frequency is 50% for a heterozygous change. On the other hand, if only a smaller subset of aligned reads indicates variation, that could result from technology bias or be a mosaicism, i.e. an individual which harbour two or more populations of genetically distinct cells as a result of postzygotic mutation. Postzygotic de novo mutations may result in somatic mosaicism (potentially causing a less severe and/or variable phenotype compared with the equivalent constitutive mutation) and/or germline mosaicism (hence enabling transmission of a pathogenic variant from an unaffected parent to his affected offspring) (Biesecker et al., 2013). To identify mosaicism, a probabilistic approach should consider deviation of the proband variant allele fraction (VAF, defined as the number of alternative reads divided by the total read depth) from a binomial distribution centred around 0.5.


igv_screenshot_variant

Figure 5. Variant visualization by IGV


The GATK variant calling pipeline first produces a genomic VCF (gVCF), whose main difference with VCF is that it records all sites in a target whether there is a variant or not, while VCF contains only information for variant sites, preparing multiple samples for joint genotyping and creation of a multi-sample VCF whose variants can undergo quality filtering in order to obtain the final set of quality-curated variants ready to be annotated.

In downstream analyses, annotations can be added to VCF files themselves or information in VCF files can be either annotated in TAB- or comma- deimited files to be visually inspected for clinical variant searching or used as input to prioritization programs.

Quality control

In-depth quality control (QC) of data generated during an NGS experiment is crucial for an accurate interpretation of the results. For example an accurate QC could help in identifying poor quality experiments, sequence contamination or genomic regions with low sequence coverage, and all these factors have a large impact on the downstream processing.

Most of the programs used during an NSG workflow do not include steps for quality control, therefore artifacts needs to be detected using ad-hod developed tools for QC. The table summarizes the main tools available at https://usegalaxy.eu for quality checking, at each step of the analysis.

NGS workflow step File format Tools for quality control
Sample to reads fastQ FastQC
Reads to alignments BAM General statistics: bam.io.bio, samtools; target coverage: Picard CollectHSMetrics; per base coverage depth: bedtools
Alignments to variants VCF vcf.io.bio

Quality control of FASTQ files

Before starting the analysis workflow, you should identify possible issues that could affect alignment and variant calling. This first step of quality control is based on the raw sequence data (fastQ) generated by the sequencer. Common issues with sequence quality can be easily addressed by further processing your original sequences to trim or remove low-quality reads. In presence of severe artefacts you should consider to repeat the experiment instead of starting the downstream analysis that will generate poor quality results, according to the rule ‘garbage in, garbage out’.

Here we’ll use the FastQC software for a standard quality check, using the two FASTQ files HighQuality_Reads.fastq and LowQuality_Reads.fastq.

FastQC is relatively easy to use. The output of FastQC consists of multiple modules analysing a specific aspect of the quality of the data. A detailed help can be found in the help manual.

The names of the modules are preceded by an icon that reflects the quality of the data, and indicates whether the results of the module are:

  • normal (green)
  • slightly abnormal (orange)
  • very unusual (red)

comment Note on FastQC interpretation

These evaluations must be taken in the context of what you are expecting from your dataset. For FastQC a normal sample includes random sequences with high diversity. If your experiment generates biased libraries (e.g. low complexity libraries) you should interpret the report with attention. In general, you should concentrate on the icons different from green and try to understand the reasons for this behaviour.

hands_on Hands-on: Compute sequence quality with FastQC

  1. Run FastQC tool on your fastq datasets HighQuality_Reads.fastq and LowQuality_Reads.fastq. You can select both datasets with the Multiple datasets option.

    tip Tip: Select multiple datasets

    1. Click on param-files Multiple datasets
    2. Select several files by keeping the Ctrl (or COMMAND) key pressed and clicking on the files of interest

    For each input file you will get two datasets, one with the raw QC statistics and another with an HTML report with figures.

  2. Using the MultiQC tool software, you can aggregate multiple raw FastQC output in one unique report. This helps in comparing multiple samples at the same time in order to quickly identify low quality samples that will be displayed as outliers.
    • “Which tool was used generate logs?”: FastQC
      • In “FastQC output”
        • “Type of FastQC output?”: Raw data
        • param-files “FastQC output”: the two RawData outputs of FastQC tool
  3. Inspect MultiQC report

For a detailed explanation of the different analysis modules of FastQC you may refer to the Quality control tutorial.

question Questions

  1. Based on the MultiQC report, check which modules highlight differences in sequence quality betwen the two datasets

Quality control of BAM files

Fast quality check with bam.iobio.io

BAM files are binary files containing information on the sequences aligned onto a reference genome. Exploring BAM files you can address several questions, e.g.::

  • which is the amount of duplicated sequences? For non PCR-free protocols, it should be < 15%. Duplicated sequences are not used in downstream analysis to identify variants, therefore should be kept at a minimum to avoid waste of reagents.
  • which is the fraction of unmapped reads? It should be < 2%. If higher, you should ask why so many reads are not properly mapped onto the reference genome. One possible reason could be sample contamination.

In Galaxy, BAM files can be explored using the bam.iobio.io web app. Leveraging on random subsampling of reads, bam.iobio.io quickly draws several quality control reports (Figure 6).

On top of each plot, clicking on the question mark you can open a window with a detailed explanation of the expected output. The number of reads sampled is shown at the top-right of the page, and can be increased by clicking on the arrow.

bam_iobio

Figure 6. BAM quality control using bam.iobio.io

hands_on Hands-on: Compute BAM quality with bam.iobio.io

  1. Run bam.iobio.io tool on a BAM dataset. To start bam.iobio.io click on the link diplay at bam.iobio.io in the dataset section. Please note that the link will be visible only for datasets with the appropriate database field set to hg19

    tip Tip: Changing Database/Build (dbkey)

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, change the Database/Build field
    • Select your desired database key from the dropdown list: hg19
    • Click the Save button

    question Questions

    1. Which is the amount of duplicated sequences?
    2. And the fraction of aligned reads?

Computation of per-base coverage depth at specific genomic intervals

Commercial next-generation sequencing platform usually provide users with analysis programs that include tools for the identification of low coverage regions (for instance, target regions that have a coverage depth lower than 20x).

The present tutorial is aimed to show how to perform a custom coverage analysis of NGS experiment(s) by using tools that are available in Galaxy.

Starting material:

  • Alignment (bam) file(s) on which you want to perform the coverage evaluation.
  • A reference bed file listing the genomic regions for which you want to obtain coverage data. If you performed a targeted sequencing experiment by using commercial kits (either custom or from the catalogue), you should already have obtained a bed file listing the target regions: it should be the file you want to use.

comment BED format specifications

BED files are tab-delimited files with one line for each genomic region. The first 3 fields (columns) are required:

  1. chromosome
  2. the starting position
  3. the ending position

Further columns may be present but are optional. Additional details may be found here: UCSC BED format specifications

Please be aware that in BED files the description of genomic regions follows the “0-start, half-open” coordinate system. Further details may be found here: the “0-based, half-open” UCSC Genome Browser Coordinate Counting Systems. Practically speaking, it means that the starting position is diminished by 1 bp with respect to the actual position. For instance, the region “chr2 50149071 50149399” in a bed file corresponds to the genomic interval from position 50149072 (included) to position 50149399 (included) on chromosome 2 (when the first base of the chromosome is defined as position 1). If you want to indicate a single base position in a bed file, it should be written like this: “chr2 50149071 50149072” .

hands_on Hands-on: Compute per base coverage depth with BEDtools

Before starting, you have to upload the files you need for the analysis, following standard Galaxy procedure.

You may use files provided as examples with this tutorial and called Panel_alignment.bam and Panel_Target_regions.bed.

Please check that uploaded file datatypes (formats) are properly recognized by selecting edit attributes (i.e. the pencil sign in correspondence of each file you can find in your history) (indicated by the red arrow in figure 1) and then the tab datatypes (the blue arrow in figure 1). If the datatype is wrong, select the correct one from the drop-down list and press the change datatype button (the green arrow in figure 1).


Figure 1 Figure 1


Once ready, you can select the tool named bedtools Compute both the depth and breadth of coverage in the Operate on Genomic Intervals section (see the red arrow in figure 2).

  1. Select the bed file listing the target regions as “file A” (blue arrow in figure 2) and the bam file(s) you want to analyze as “file B” (green arrow in figure 2) (they should be listed in the drop-down menu if they have the correct format). You may analyze one or more bam files in a single run.
  2. If you want to analyze two or more .bam files, you can further choose if you want all the results in a single file or one output file per input “file B” by selecting the desired option under the Combined or separate output files menu.
  3. Select “Yes” for the option Report the depth at each position in each A feature (yellow arrow in figure 2) and check that all the other options are set to “No”.
  4. Star (Execute) the analysis.

    Figure 2 Figure 2


Output file, which will be called “coverage_depth.bed” from now on, will contain all the fields of the original target_regions.bed file plus two further columns:

  1. the first value indicates a specific base within the reported genomic interval. For instance, if the genomic interval described by the first 3 field is “chr2 50149071 50149072” and the first new field reports the number 1000, it means that the coverage value refers to nucleotide 50150071 (i.e.: 50149071 + 1000) on chromosome 2;
  2. the second value indicates the depth of coverage for the defined base.

hands_on Hands-on: Sort files

Since entries in the “coverage_depth.bed” may not be in the desired order, you can sort it by genomic positions. For this purpose you may want to use the Sort tool in the Text Manipulation section (check out the blue arrow in figure 3).

You may sequentially sort on different columns:

  1. first you can sort by chromosome by selecting column 1 (green arrow in figure 3) in “ascending order” and selecting the “flavor” Natural/Version sort (-V), which allows for sorting chromosomes in their “natural” order (with alphabetical order chr10 will be right after chr1, chr2 after chr19 and so on);
  2. after inserting a new column section (red arrow in figure 3), you can sort by column 2 in “ascending order” with Fast numeric sort (-n);
  3. after inserting a further column section, you can sort by column 3 in “ascending order” with Fast numeric sort (-n);
  4. after inserting a final column section, you can sort by column 3 in “ascending order” with Fast numeric sort (-n).

Figure 3 Figure 3


The obtained output file, which will be called “sorted_coverage_depth.bed” from now on, will be sorted first by chromosome, then by starting position, by ending position and by the actual position of the specific base in the considered genomic interval.

hands_on Hands-on: Remove duplicate rows

If your file contains duplicated lines you may want to remove them for further processing. For this purpose you can use the Unique lines assuming sorted input file tool in the Text Manipulation section.

hands_on Hands-on: Some manipulation of the bed file

You may follow the following steps to manipulate the “sorted_coverage_depth.bed” and to obtain a bed file listing the exact base position to which each coverage value is referred. For instance instead of having “chr2 50149071 50149399 NRXN1 1 2335” in the first row of your file, you will get “chr2 50149071 50149072 NRXN1 2335”.

These steps will add further columns at the end of each line defining the base position with the “0-start, half-open” coordinate system.

  1. Select the Compute an expression on every row tool in the Text Manipulation section (indicated by the blue arrow in figure 4);
  2. add the following expression “c2+c5-1” to obtain the sum of the values in columns 2 and 5 minus 1 (it will be used as the new starting position in the final file);
  3. select the file “sorted_coverage_depth.bed”;
  4. select “yes” to round Results? (green arrow in figure 4);
  5. execute;
  6. you can rename the output as “temp1_coverage_depth.bed”;
  7. select the Compute an expression on every row tool in the Text Manipulation section;
  8. add the following expression “c2+c5” to obtain the sum of the values in columns 2 and 5 (it will be used as the new ending position in the final file). You can also use the expression ;
  9. select the file “temp1_coverage_depth.bed”;
  10. select “yes” to round Results?;
  11. execute;
  12. you can rename the output as “temp2_coverage_depth.bed”;
  13. select the Table Compute tool in the Text Manipulation section (indicated by the blue arrow in figure 5);
  14. select the file “temp2_coverage_depth.bed”;
  15. select the option Drop, keep or duplicate rows and columns from the drop-down menu Type of table operation (indicated by the green arrow in figure 5);
  16. fill the field List of columns to select with “1,7,8,4,6” (the red arrow in figure 5);
  17. unselect all the other options;
  18. execute;
  19. set the output file datatype to bed;
  20. you can rename the output as “final_coverage_depth.bed”.

Figure 4 Figure 4

Figure 5 Figure 5


hands_on Hands-on: Select positions with low coverage depth

The following procedure can be used to obtain a bed file listing base positions with a coverage depth lower than a certain threshold (for instance 20x).

  1. Select the Filter tool in the Filter and Sort section (indicated by the blue arrow in figure 6);
  2. select the file “final_coverage_depth.bed”;
  3. add the following expression “c5<20” to filter all positions with a coverage depth lower than (green arrow in figure 6)(“c5” stands for the fifth column, in this case reporting the coverage depth);
  4. execute.

Figure 6 Figure 6


The output file, which will be called “low_coverage_depth.bed” from now on, will only list all the positions with a depth lower than 20x.

hands_on Hands-on: Merge low coverage regions

If you want to merge the positions with low coverage depth in larger genomic intervals to be used for further analyses (i.e.: Sanger sequencing of regions not covered by your NGS experiment), you may want to use the bedtools MergeBED tool in the Operate on Genomic Intervals section (see the blue arrow in figure 7).

  1. Select the file “low_coverage_depth.bed”;
  2. set the maximum distance between features (i.e.: the different positions listed in your file) allowed for features to be merged (green arrow in figure 7): if it is 0 only overlapping and/or book-ended features are merged, while if it is set to 10 (or any other different positive integer of your choice), features at the maximum distance of 10 bases will be merged;
  3. if you want, you may apply some operations to certain columns to get further information in your output file. For instance you may:
    1. click on “Insert Applying operations to columns from merged intervals” (red arrow in figure 7),
    2. specify the column on which you want to perform the operation (in this case column 5),
    3. and select the operation from the drop-down list(in this case “Min”, which calculates the minimum value of coverage depth among all the positions that will be merged in a single interval) (yellow arrow in figure 7);
  4. you may add as many operations as you need. In this example we will also calculate the maximum value of coverage depth;
  5. execute.

Figure 7 Figure 7


The output file will have the following fields (columns): chromosome, starting and ending positions of low coverage regions, the minimum and the maximum coverage depth in each region.

warning BED files may have different columns

Please be aware that the columns to use for calculations may be different compared to the example here considered, depending on the amount of columns of your bed files.

warning Preview of BED files

Please be aware that the Galaxy preview of your file shows a header row that does not properly define columns in your files (it is just a standard header for the UCSC bed format).

Final notes

The procedures listed above are to be taken as examples of the possible operations that can be performed on bed files with bedtools (you may check out their website to get further information: BEDtools) ad text manipulation tools available on Galaxy.

Furthermore, please be aware that the tool bedtools Compute both the depth and breadth of coverage does not perform any filtering based on read quality: if your are interested in that aspect you may want to rely on different tools.

Variant calling and classification

After the generation of a high-quality set of mapped read pairs, we can proceed to call different classes of DNA variants. To this aim, the tools HaplotypeCaller and MuTect2 from GATK toolkit are a dedicated solution for DNA variant identification at germinal- and somatic-level. They can:

  • Determine haplotypes by local assembly of the genomic regions in which the samples being analyzed show substantial evidence of variation relative to the reference;
  • Evaluate the evidence for haplotypes and variant alleles;
  • Assigning per-sample genotypes.

hands_on Hands-on: Variant calling.

You may use files provided as examples with this tutorial and called Panel_alignment.bam and Panel_Target_regions.bed.

  1. Run HaplotypeCaller tool restricting the search space on target regions with “-L” option to reduce computational burden.

In case of analyzing a single sample:

gatk HaplotypeCaller -I Panel_alignment.bam -O Sample1.all_exons.hg19.vcf -L Panel_target_regions.bed -R HSapiensReference_genome_hg19.fasta

In case of analyzing a cohort (e.g. family) of samples, the most efficient way to identify variants is a multi-step process, running HaplotypeCaller per-sample to generate many intermediate gVCF files, merge them, and then calling genotypes on the whole group, based on the alleles that were observed at each site:

gatk HaplotypeCaller -I Panel_alignment.bam -O Sample1.all_exons.hg19.vcf -R HSapiensReference_genome_hg19.fasta -L Panel_target_regions.bed -ERC GVCF

To obtain genotypes for all the cohort provide a combined multi-sample GVCF and then use it to calculate likelihoods:

  • gatk CombineGVCFs --variant Sample1.all_exons.hg19.vcf --variant Sample2.all_exons.hg19.vcf -O cohort.all_exons.hg19.g.vcf -L Panel_target_regions.bed -R HSapiensReference_genome_hg19.fasta

  • gatk GenotypeGVCFs -V cohort.all_exons.hg19.g.vcf -O cohort_genotyped.all.exons.vcf -L Panel_target_regions.bed -R HSapiensReference_genome_hg19.fasta

The genotyped SNV/INDELs are then stored in a VCF file to be functionally annotated.

  1. Run Mutect2 tool restricting the search space on target regions with “-L” option to reduce computational burden. The first step is needed to create an internal database of controls (i.e. Panel Of Normals - PoN) to reduce bias for somatic calls. It runs on a single sample at time:
  • gatk Mutect2 -R HSapiensReference_genome_hg19.fasta -L Panel_target_regions.bed -I Panel_alignment_normal1.bam -O normal_genotyped1.vcf
  • gatk Mutect2 -R HSapiensReference_genome_hg19.fasta -L Panel_target_regions.bed -I Panel_alignment_normal2.bam -O normal_genotyped2.vcf

Then use GATK’s CreateSomaticPanelOfNormals tool to generate the PoN:

  • gatk GenomicsDBImport -L Panel_target_regions.bed -R HSapiensReference_genome_hg19.fasta --genomicsdb-workspace-path PoN_db -V normal_genotyped1.vcf -V normal_genotyped2.vcf
  • gatk CreateSomaticPanelOfNormals -R HSapiensReference_genome_hg19.fasta -V gendb://PoN_db -O panel_of_normals.vcf

    comment Note

    The –genomicsdb-workspace-path must point to a non-existent or empty directory.

Then, to effectively call somatic mutations, we can use variants contained in the PoN and/or other public repositories (e.g. by means of the option –germline-resource, using a VCF file containing frequencies of germline variants in the general population) to exclude germline variation. Finally, to properly classify somatic variants, we apply FilterMutectCalls filtering, which produces the final subset annotated VCF file. To this aim, we can run the following commands:

  • gatk Mutect2 -R HSapiensReference_genome_hg19.fasta -I Panel_alignment.bam --germline-resource af-only-gnomad.vcf --panel-of-normals panel_of_normals.vcf -O somatic_genotyped_unfiltered.vcf

  • gatk FilterMutectCalls -R HSapiensReference_genome_hg19.fasta -V somatic_genotyped_unfiltered.vcf -O somatic_genotyped_filtered.vcf

Variant annotation

Once called, variants (SNPs and InDels) need to be annotated.

We want to know for example if a variant is located in a gene, if it’s in the coding portion of that gene, if it causes an aminoacid substitution, if that substitution is deleterious for the encoded protein function.

Variant annotation is the process of attaching technical and/or biological information from multiple available source (e.g. public databases) to variants


Gene model

The choice of gene model is essential for variant downstream variant annotation: it describe genomic positions of genes and each exon-intron exact locations

Different gene models can give different annotations: BRCA1

Figure 1. Variant indicated by the red dashed line can be annotated as intronic or exonic (on one of the UCSC transcript variants), depending on the adopted gene model:


Sequence variant nomenclature

Variant nomenclature should be described univocally:

Variant class

Population sequencing db

Variant-disease/gene-disease db

Annotation Software and tools


Annotation and filtering with wANNOVAR

The web tool wANNOVAR allows for rapid annotation of your variants and for some basic filtering steps to find disease genes. It is based on its command line counterpart ANNOVAR, but it is more user-friendly since it does not require any programming skills.

The output consist in tabular text files that can be easily manipulated with Excel or other spreadsheet programs.

The annotation is performed against some of the most commonly used databases: RefSeq, UCSC Known, ENSEMBL/Genecode, dbSNP, ClinVar, 1000genomes, ExAC, ESP6500, gnomAD (minor allele frequencies in different populations are reported) and various precalculated prediction scores for any possible single nucleotide variant in coding regions (see dbNSFP).

The gene-based annotation results in a single row for each input variant: only the most deleterious consequence is reported (i.e.: if a certain variant may result to be missense for one transcript and nonsense for a second transcript, only the latter consequence will be reported).

Unfortunately, unlike the command line version, wANNOVAR does neither allow for the use of custom annotation databases, nor for the selection of different pubblicly available databases.

To annotate your file with wANNOVAR you need to provide your email address, to be notified when the annotation is complete, and just upload your input file (or paste a series of variant in the designated field). Results are usually ready within minutes. Figure 1

You will get both csv files or txt files (which can be saved as they are by clicking on the specific link, right-clicking in any point of the page and selecting Save as). They can be both opened with Excel or other spreadsheet programs (concerning csv files, please ensure that comma are set as default list separator/delimiter in your version of Excel).

You will also get both exome summary results (only conding variants are included) and genome summary results (all variants included).

You can also provide a list of Disease or Phenotype Terms that the program can use for filtering your results (only for single sample vcf files). Figure 2

Finally, there are some Parameter Settings that can be modified:

  • Result duration: it can now only be set to “1 day”, since your files will be automatically removed after 24 hours.
  • Reference genome: you can choose between hg19 (GRCh37) and hg38 (GRCh38).
  • Input Format: you can upload not only vcf files, but also other kinds of variant files.
  • Gene Definition: the database you want to use for gene function annotation. Three oprtions are available: RefSeq, UCSC Known, ENSEMBL/Gencode
  • Individual analysis: the “Individual analysis” option allows you to perform further filtering steps (based on the Disease/Phenotype terms or the Disease Model) on a single sample (if you upload a multisample vcf only the first sample will be considered); the “All Annotaions” option will annotate all variants in your multisample vcf, maintaining the original columns of your vcf as the last columns of your output file.
  • Disease Model: this options allows for some basic filtering of your variants based on the expected mechanism of inheritance. In mainly consider frequencies, sample genotypes and consequences at level of genic function. FIltering step are summarized among the results and they are only performed on a single sample: the program does not perform any multisample evaluation (i.e.: variant segregation in a trio) and cannot classify any variant as de novo even if you provide a multisample vcf with parental genotypes.

Clinical databases for further manual variant annotation

Once you have obtained a file with the annotation of your variants, you might find useful to annotate also the involved genes, in order to know, for instance, the list of diseases that may be associated with them.

Some databases that can the jb are the following:

  1. The gene2phenotype dataset (G2P) integrates data on genes, variants and phenotypes for example relating to developmental disorders. In the “Download” section you will find both databases of genes related to cancer and to developmental disorders. Those files report for each gene listed: the OMIM number, the associated disease name, the disease OMIM number, the disease category (you can fing more details in the “Terminology” section), whether the disease is caused by biallelic or monoallelic variants (“allelic requirement”), the expected category of variant to be causative of the disease, and a few other details.
  2. In the “Download” section of the OMIM database, if you register for research and educational use, you may obtain different lists of OMIM genes and their associated phenotypes.
  3. Among the files available for download from the gnomAD database, you may get per-gene constraint scores (for further details, please check the paper by the Exome Aggregation Consortium on “Nature. 2016 Aug 18; 536(7616):285–291.”). Those score may indicate if a specific gene is expected to be intolerant to loss-of-function variants (pLI) (haploinsufficiency), or if it is predicted to be associate to recessive diseases.

In the end, you can add these annotations to your wANNOVAR files by using the VLOOKUP function in Excel.

Variant prioritization

Once annotated, variants need to be filtered and prioritized

  • WES: Tens of thousands
  • WGS: Millions

No universal filters, they depend on the experimental features

Variant impact

First of all you usually want to filter variants by consequence on the encoded protein, keeping those which have an higher impact on protein:

  • Missense
  • Nonsense
  • Splice sites
  • Frameshift indels
  • Inframe indels

Variant frequency

  • Common variants are unlikely associated with a clinical condition
  • A rare variant will probably have a higher functional effect on the protein
  • Frequency cut-off have to be customized on each different case
  • Typical cut-offs: 1% - 0.1%
  • Allele frequencies may differ a lot between different populations

Variant effect prediction Tools

  • Tools that predict consequences of amino acid substitutions on protein function
  • They give a score and/or a prediction in terms of “Tolerated”, “Deleterious” (SIFT) or “Probably Damaging”, “Possibly Damaging”, “Benign” (Polyphen2)

    • fitCons
    • GERP++
    • SIFT
    • PolyPhen2
    • CADD
    • DANN
    • Condel
    • fathmm
    • MutationTaster
    • MutationAssessor
    • REVEL

ACMG/AMP 2015 guidelines

The American College of Medical Genetics and the Association for Molecular Pathology published guidelines for the interpretation of sequence variants in May of 2015 (Richards S. et al, 2015). This report describes updated standards and guidelines for classifying sequence variants by using criteria informed by expert opinion and experience

  • 28 evaluation criteria for the clinical interpretation of variants. Criteria falls into 3 sets:
    • pathogenic/likely pathogenic (P/LP)
    • benign/likely benign (B/LB)
    • variant of unknown significance (VUS)
  • Intervar: software for automatical interpretation of the 28 criteria. Two major steps:
  • automatical interpretation by 28 criteria
  • manual adjustment to re-interpret the clinical significance

Prioritization

Phenotype-based prioritization tools are methods working by comparing the phenotypes of a patient with gene-phenotype known associations.

  • Phenolyzer: Phenotype Based Gene Analyzer, a tool to prioritize genes based on user-specific disease/phenotype terms

hands_on Hands-on: Variant prioritization

Using knowledge gained on Genomic databases and variant annotation section, try to annotate, filter and prioritize an example exome variant data, using two disease terms

  • Download file Sample1.all_exons.hg19.vcf from the dataset, as learned in the Home section

  • Go to wANNOVAR

  • Use the vcf file as input file and hearing loss and deafness autosomal recessive, as disease terms to prioritize results

  • Choose rare recessive Mendelian disease as Disease Model in the Parameter Settings section

  • Provide an istitutional email address and submit the Job

  • wait for results

    In the results page you can navigate and download results. Click Show in the Network Visualization section to see Phenolyzer prioritization results

CNV detection from targeted sequencing data

  • Copy Number Variants (CNVs) are imbalances in the copy number of the genomic material that result in either DNA deletions (copy loss) or duplications
  • CNVs can cause/predispose to human diseases by altering gene structure/dosage or by position effect
  • CNV size ranges from 50 bp to megabases
  • Classical methods to identify CNVs use array-based technologies (SNP/CGH)
  • Computational approaches have been developed to identify CNVs in targeted sequencing data from hybrid capture experiments

Computational approaches

There are four main methods for CNV identification from short-read +NGS data (see figure below):

  • Read Count (RC)
  • Read Pair (RP)
  • Split Read (SR)
  • De Novo Assembly (AS)

  • RP and SR require continuous coverage of the CNV region or reads encompassing CNV breakpoints, as in whole genome sequencing. The sparse nature and small size of exonic targets hamper the application of these methods to targeted sequencing.
  • RC is the best suited method for CNV detection from whole exomes or gene panels where:
  • deletions appear as exonic targets devoid of reads
  • duplications appear as exonic targets characterized by excess of coverage

CNV detection methods

Figure 1. Methods for detection of CNVs in short read NGS data (adapted from Tattini et al., 2015)


RC method and data normalization

In targeted sequencing, a method to study DNA copy number variation by RC (as implemented in EXCAVATOR tool, Magi et al., 2013)) is to consider the exon mean read count (EMRC):

EMRC = RCe/Le

where RCe is the number of reads aligned to a target genomic region e and Le is the size of that same genomic region in base pairs (Magi et al., 2013)). Three major bias sources are known to affect EMRC dramatically in targeted sequencing data:

  • local GC content percentage
  • genomic mappability
  • target region size

These biases contribute to non uniform read depth across target regions and, together with the sparse target nature, challenge the applicability of RC methods to targeted data. As shown in Figure 2 (left panel), in single-sample data the EMRC distributions of genomic regions characterized by different copy numbers largely overlap, revealing poor CNV prediction capability.

EMRC ratio between two samples can be used as a normalization procedure. The effect of EMRC ratio-based normalization is clear in Figure 2 (right panel) as a markedly improved correspondece between the predicted and the real copy number states.


Data Normalization_1

Figure 2. Effect of EMRC ratio on DNA copy number prediction (adapted from Magi et al., 2013)


Similarly FPKM, a normalized measure of read depth implemented in ExomeDepth tool (Plagnol et al., 2012), is affected by extensive exon–exon variability but comparison between pairs of exome datasets demonstrates high correlation level of the normalized read count data across samples making it possibile to use one or more combined exomes as reference set to base the CNV inference on (Plagnol et al., 2012).


Data Normalization_2

Figure 3. Correlation of the normalized read count data between samples (from Plagnol et al., 2012)


CNV detection accuracy

Strong correlation is observed between Affymetrix array-SNP and exome-derived data for CNVs >1 Mb (Figure 4, right panel), while including CNVs of any size dramatically decreases the correlation level (Figure 4, left panel). This can be explained by the different distribution of exons and SNP probes throughout the genome. Candidate CNV regions as identified by EXCAVATOR contain a comparable number of exons and SNP probes when they are > 1 Mb (R=0.8), while regions <100 Kb do not (R=-0.02). Accordingly, Krumm et al., 2012 report 94% precision in detecting CNVs across three or more exons.


Correlation_Exome_Affymetrix

Figure 4. Correlation between array-SNP and exome-derived CNVs including all (left panel) or > 1 Mb CNVs (adapted from Magi et al., 2013)


To increase chances to detect CNVs encompassing few exons or in non-coding regions from exome data, D’Aurizio et al., 2016 have proposed an extension to EXCAVATOR to exploit off-target read count (EXCAVATOR2). Similar approaches taking advantage of off-target reads have been described by Bellos et al., 2014 or Talevich et al., 2016.

Tools for CNV detection from gene panels or exome data

A number of tools or pipelines implementing modified versions of previously published tools have been reported to detect single-exon CNVs in clinical gene panels:

In addition to the above-mentioned methods, many tools have been developed that detect CNVs from exomes. Evaluation of these tools is not straightforward as there is lack of a gold standard for comparison. As a consequence, there is no consensus level on pipelines as high as for single nucleotide variants. Zare et al., 2017 have reviewed this topic with focus on cancer, reporting poor overlap of different tools and added challenges for somatic variant calling.


hands_on Hands-on: CNV calling with ExomeDepth

ExomeDepth uses pairs of case and control exomes to identify copy number imbalances in case(s) compared to control(s). Here we provide a small data set including a pair of case/control BAM files and an exome target BED file restricted to a specific polymorphic region on chromosome 2q.

  • CNV_case.bam.
  • CNV_control.bam.
  • CNV_TruSeq_Chr2.bam

    Your aim is to identify a large polymorphic deletion in case.

Regions of Homozygosity

  • Runs of Homozygosity (ROHs) are sizeable stretches of consecutive homozygous markers encompassing genomic regions where the two haplotypes are identical
  • Haplotypes in ROHs can underlie identity either by state (IBS) or by descent (IBD). IBD occurs when two haplotypes originate from a common ancestor (Figure 1), a condition whcih we refer to as autozygosity
  • Autozygosity in an individual is the hallmark of high levels of genomic inbreeding (gF), often occurring in the offspring from consanguineous unions where parents are related as second-degree cousins or closer
  • The most well-known medical impact of parental consanguinity is the increased risk of rare autosomal recessive diseases in the progeny, with excess risk inversely proportional to the disease-allele frequency (Bittles, 2001)

homozygosity_mapping Figure 1. Schematic of haplotype flow along a consanguineous pedigrees to form autozygous ROHs in the progeny (from McQuillan et al., 2008)


Homozygosity mapping

  • We refer to homozygosity mapping (Lander and Botstein, 1987) as an approach that infers autozygosity from the detection of long ROHs to map genes associated with recessive diseases
  • A classical strategy to identify ROHs is based on SNP-array data and analysis with PLINK
  • The combination of homozygosity mapping with exome sequencing has boosted genomic research and diagnosis of recessive diseases in recent years (Alazami et al., 2015; Monies et al., 2017)
  • ROHs may be of clinical interest also since they can unmask uniparental isodisomy
  • Computational approaches have been developed to identify ROHs directly in exome data, allowing simultaneous detection of variants and surrounding ROHs from the same datasets.

Computational approaches

As for CNVs, the sparse nature and small size of exonic targets are a challenge for the identification of continuous strethces of homozygous markers throughout the genome. This may affect the performance of tools originally tailored to SNPs when applied to exome data.

In Figure 2 a synthetic overview is given of algorithms, input data and output file types in bioinformatic tools for exome-based ROH detection. ROHs are divided in three size classes reflecting different mechanisms that have shaped them (according to Pemberton et al., 2012).

Usually, long ROH (approximately >1.5 Mb) arise as a result of close parental consanguinity, but also short-medium ROHs can be of medical interest in populations as involved in disease susceptibility, natural selection and founder effects (Ceballos et al., 2018).


roh_exome_methods Figure 2. Summary of the tools for ROH detection from exome data (from Pippucci et al., 2014)


H3M2

H3M2 (Magi et al., 2014) is based on an heterogeneous Hidden Markov Model (HMM) that incorporates inter-marker distances to detect ROHs from exome data.

H3M2 calculates B-allele frequencies of a set of polymorphic sites throughout the exome as the ratio between allele B counts and the total read count at site i:

BAFi = Nb/N

H3M2 retrieves BAFi directly from BAM files to predict the heterozygous/homozygous genotype state at each polymorphic position i:

  • when BAFi ~ 0 the predicted genotype is homozygous reference
  • when BAFi ~ 0.5 the predicted genotype is heterozygous
  • when BAFi ~ 1 the predicted genotype is homozygous alternative

H3M2 models BAF data by means of the HMM algorithm to discriminate between regions of homozygosity and non-homozygosity according to the BAF distribution along the genome (Figure 3).


h3m2_baf Figure 3. BAF data distribution Panels a, b and c show the distributions of BAF values against the genotype calls generated by the HapMap consortium on SNP-array data ( a ), the genotype calls made by SAMtools ( b ) and the genotype calls made by GATK ( c ). For each genotype caller, the distribution of BAF values is reported for homozygous reference calls (HMr), heterozygous calls (HT) and homozygous alternative calls (HMa). R is the Pearson correlation coefficient. Panels d–g show the distribution of BAF values in all the regions of the genome ( d ), in heterozygous regions ( e ), in homozygous regions ( f ) and in the X chromosome of male individuals ( g ). For each panel, the main plot reports the zoomed histogram, the left subplot shows the BAF values against genomic positions, whereas the right subplot shows the entire histogram of BAF values. (from Magi et al., 2014)

Contributors

  • Tommaso Pippucci - Sant’Orsola-Malpighi University Hospital, Bologna, Italy
  • Alessandro Bruselles - Istituto Superiore di Sanità, Rome, Italy
  • Andrea Ciolfi - Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy
  • Gianmauro Cuccuru - Albert Ludwigs University, Freiburg, Germany
  • Giuseppe Marangi - Institute of Genomic Medicine, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Roma, Italy
  • Paolo Uva - Centro di Ricerca, Sviluppo e Studi Superiori in Sardegna (CRS4), Pula, Cagliari, Italy

keypoints Key points

  • Focus on clinical interpretation of variants.

  • Provides real uses cases.

  • Use public tools and free annotation servers.

Citing this Tutorial

  1. Alessandro Bruselles, Andrea Ciolfi, Gianmauro Cuccuru, Giuseppe Marangi, Paolo Uva, Tommaso Pippucci, 2020 Data analysis and interpretation for clinical genomics (Galaxy Training Materials). /clinical_genomics/ Online; accessed TODAY
  2. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012

details BibTeX

@misc{-,
    author = "Alessandro Bruselles and Andrea Ciolfi and Gianmauro Cuccuru and Giuseppe Marangi and Paolo Uva and Tommaso Pippucci",
    title = "Data analysis and interpretation for clinical genomics (Galaxy Training Materials)",
    year = "2020",
    month = "01",
    day = "24"
    url = "\url{/clinical_genomics/}",
    note = "[Online; accessed TODAY]"
}
@article{Batut_2018,
        doi = {10.1016/j.cels.2018.05.012},
        url = {https://doi.org/10.1016%2Fj.cels.2018.05.012},
        year = 2018,
        month = {jun},
        publisher = {Elsevier {BV}},
        volume = {6},
        number = {6},
        pages = {752--758.e1},
        author = {B{\'{e}}r{\'{e}}nice Batut and Saskia Hiltemann and Andrea Bagnacani and Dannon Baker and Vivek Bhardwaj and Clemens Blank and Anthony Bretaudeau and Loraine Brillet-Gu{\'{e}}guen and Martin {\v{C}}ech and John Chilton and Dave Clements and Olivia Doppelt-Azeroual and Anika Erxleben and Mallory Ann Freeberg and Simon Gladman and Youri Hoogstrate and Hans-Rudolf Hotz and Torsten Houwaart and Pratik Jagtap and Delphine Larivi{\`{e}}re and Gildas Le Corguill{\'{e}} and Thomas Manke and Fabien Mareuil and Fidel Ram{\'{\i}}rez and Devon Ryan and Florian Christoph Sigloch and Nicola Soranzo and Joachim Wolff and Pavankumar Videm and Markus Wolfien and Aisanjiang Wubuli and Dilmurat Yusuf and James Taylor and Rolf Backofen and Anton Nekrutenko and Björn Grüning},
        title = {Community-Driven Data Analysis Training for Biology},
        journal = {Cell Systems}
}
                    

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