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• Introduction
  • The concept of Biomedical Genomics Analysis
  • System requirements
  • Contact information
• Data import and export
  • BGI/MGI Sequencing Import
  • Illumina Custom Reads
• Reference Data Management
  • QIAGEN Sets
• SARS-CoV-2 workflows
  • Identify ARTIC V3 SARS-CoV-2 Low Frequency and Shared Variants (Illumina)
  • Identify QIAseq SARS-CoV-2 Low Frequency and Shared Variants (Illumina)
  • Identify Ion AmpliSeq SARS-CoV-2 Low Frequency and Shared Variants (Ion Torrent)
  • SARS-CoV-2 workflow output
    • Summary outputs
    • Sample specific outputs
• QIAseq Panel Analysis
  • The Analyze QIAseq Panels guide
    • Import your reads
    • Start a QIAseq panel analysis
    • Upload to QCI Interpret
  • How to create a custom panel analysis workflow
    • Import QIAGEN Primers
  • Create a custom Trim adapter list (optional)
  • Unique Molecular Indexes (UMIs)
    • UMI group sizes
• Targeted DNA
  • The Identify QIAseq DNA Variants ready-to-use workflows
    • Output from the Identify QIAseq DNA Variants workflows
    • Quality Control for the Identify QIAseq DNA Variants workflow
    • Identify QIAseq DNA Somatic and Germline Variants from Tumor Normal Pair (Illumina)
    • Output from the Identify QIAseq DNA Somatic and Germline Variants from Tumor Normal Pair (Illumina) workflow
  • Create QIAseq DNA CNV Control Mapping workflows
    • Output from the Create QIAseq DNA CNV Control Samples workflow
  • The Identify TMB Status ready-to-use workflows
    • Output from the Identify TMB Status workflow
  • The Detect QIAseq MSI Status ready-to-use workflow
    • Output of the Detect QIAseq MSI Status workflow
  • Detect MSI Status with Baseline Creation ready-to-use workflow
  • Targeted DNA tools detailed description
    • Annotate Structural Variants
    • Convert Annotation Track Coordinates
    • Calculate TMB Score
    • Detect MSI Status
    • Extract Reads Matching Primers
    • Generate MSI Baseline
    • Refine Read Mapping
    • Remove and Annotate with Unique Molecular Index
    • Calculate Unique Molecular Index Groups
    • Create UMI Reads from Grouped Reads
    • Create UMI Reads from Reads
    • Remove Ligation Artifacts
    • Trim Primers of Mapped Reads
    • Identify Mispriming Events
    • Annotate Variants with Unique Molecular Index Info
    • Prepare Guidance Variant Track
    • Import Gene-Pseudogene Table
• Targeted RNAscan
  • The Detect QIAseq RNAscan Fusions ready-to-use workflow
    • Output from the Detect QIAseq RNAscan Fusions workflow
  • The Perform QIAseq RNA Fusion XP Analysis ready-to-use workflows
    • Output from the Perform QIAseq RNA Fusion XP Analysis workflow
  • Interpretation of fusion results
  • Targeted RNAscan tools detailed description
    • Detect and Refine Fusion Genes
    • Annotate Fusions with Known Fusion Information
    • QC for RNAscan Panels
    • Annotate RNA Variants
    • Import Known Fusion Information Track
• Immune Repertoire
  • Perform QIAseq Immune Repertoire Analysis ready-to-use workflow
    • Output from the Perform QIAseq Immune Repertoire Analysis workflow
  • QIAseq Immune Repertoire Expert Tools
    • Import VDJtools Clonotypes
    • Immune Repertoire Analysis
    • Compare Immune Repertoires
• Targeted Multimodal
  • Perform QIAseq Multimodal Analysis (Illumina) ready-to-use workflow
    • Output from the Perform QIAseq Multimodal Analysis (Illumina)
  • Perform QIAseq Multimodal Analysis with TMB and MSI (Illumina) ready-to-use workflow
    • Output from the Perform QIAseq Multimodal Analysis with TMB and MSI (Illumina)
  • Running the workflows in batch using Metadata
• Targeted RNA
  • The Quantify QIAseq RNA Expression ready-to-use workflow
    • Output from the Quantify QIAseq RNA Expression workflow
  • Targeted RNA tools detailed description
    • The Quantify QIAseq RNA tool
• UPX 3'
  • Demultiplex QIAseq UPX 3' reads
  • The Quantify QIAseq UPX 3' ready-to-use workflow
    • Output from the Quantify QIAseq UPX 3' workflow
• Exome
  • Identify QIAseq Exome Germline Variants
    • Output from the Identify QIAseq Exome Germline Variants ready-to-use workflow
  • Create QIAseq Exome CNV Control Mapping
    • Output from the Create QIAseq Exome CNV Control Mapping workflow
  • Identify QIAseq Exome Causal Inherited Variants in Trio
    • Output from the Identify QIAseq Exome Causal Inherited Variants in Trio ready-to-use workflow
• Targeted Methyl
  • The Detect QIAseq Methylation ready-to-use workflow
    • Output from the Detect QIAseq Methylation workflow
  • Targeted Methyl associated tools
    • Finding differentially methylated regions
    • Create Methylation Level Heat Map
• QCI Interpret Integration
  • Upload to QCI Interpret
  • Prepare QCI Interpret Upload
  • Upload Prepared QCI Interpret Report
• QIAseq miRNA Analysis
  • QIAseq miRNA Quantification
    • QIAseq miRNA Quantification outputs
    • Naming isomiRs
  • QIAseq miRNA Differential Expression
  • QIAseq miRNA Expert Tools
    • Create UMI Reads for miRNA
• TruSight Oncology 500
  • The Perform TSO500 DNA Analysis (Illumina) workflow
    • Output from the Perform TSO500 DNA Analysis
  • The Perform TSO500 RNA Analysis (Illumina) workflow
    • Output from the Perform TSO500 RNA Analysis
• QIAGEN GeneRead DNA Panel Analysis
  • The QIAGEN GeneRead Panel Analysis workflow
    • Output from the QIAGEN GeneRead Panel Analysis
  • Trim Primers and their Dimers from Mapping
• Ready-to-use workflows descriptions and guidelines
  • General Workflow
  • Somatic Cancer
  • Hereditary Disease
• Getting started
  • Import sequencing data
  • Prepare Raw Data
• Whole genome sequencing (WGS)
  • General Workflows (WGS)
    • Annotate Variants (WGS)
    • Identify Known Variants in One Sample (WGS)
  • Somatic Cancer (WGS)
    • Filter Somatic Variants (WGS)
    • Identify Somatic Variants from Tumor Normal Pair (WGS)
    • Identify Variants (WGS)
  • Hereditary Disease (WGS)
    • Filter Causal Variants (WGS-HD)
    • Identify Causal Inherited Variants in Family of Four (WGS)
    • Identify Causal Inherited Variants in Trio (WGS)
    • Identify Rare Disease Causing Mutations in Family of Four (WGS)
    • Identify Rare Disease Causing Mutations in Trio (WGS)
    • Identify Variants (WGS-HD)
• Whole exome sequencing (WES)
  • General Workflows (WES)
    • Annotate Variants (WES)
    • Annotate Variants with Effect Scores (WES)
    • Identify Known Variants in One Sample (WES)
  • Somatic Cancer (WES)
    • Filter Somatic Variants (WES)
    • Identify Somatic Variants from Tumor Normal Pair (WES)
    • Identify Variants (WES)
    • Identify and Annotate Variants (WES)
  • Hereditary Disease (WES)
    • Filter Causal Variants (WES-HD)
    • Identify Causal Inherited Variants in Family of Four (WES)
    • Identify Causal Inherited Variants in Trio (WES)
    • Identify Rare Disease Causing Mutations in Family of Four (WES)
    • Identify Rare Disease Causing Mutations in Trio (WES)
    • Identify Variants (WES-HD)
    • Identify and Annotate Variants (WES-HD)
• Targeted amplicon sequencing (TAS)
  • General Workflows (TAS)
    • Annotate Variants (TAS)
    • Identify Known Variants in One Sample (TAS)
  • Somatic Cancer (TAS)
    • Filter Somatic Variants (TAS)
    • Identify Somatic Variants from Tumor Normal Pair (TAS)
    • Identify Variants (TAS)
    • Identify and Annotate Variants (TAS)
  • Hereditary Disease (TAS)
    • Filter Causal Variants (TAS-HD)
    • Identify Causal Inherited Variants in Family of Four (TAS)
    • Identify Causal Inherited Variants in Trio (TAS)
    • Identify Rare Disease Causing Mutations in Family of Four (TAS)
    • Identify Rare Disease Causing Mutations in Trio (TAS)
    • Identify Variants (TAS-HD)
    • Identify and Annotate Variants (TAS-HD)
• Whole Transcriptome Sequencing (WTS)
  • Annotate Variants (WTS)
  • Compare Variants in DNA and RNA
  • Identify Candidate Variants and Genes from Tumor Normal Pair
  • Identify Variants and Add Expression Values
  • Identify and Annotate Differentially Expressed Genes and Pathways
• General tools
  • Create Consensus Sequences from Variants
  • CNV and LOH Detection
    • Region-level CNV track (Region CNVs)
    • Target-level CNV track (Target CNVs)
    • Gene-level annotation track (Gene CNVs)
    • Loss-of-heterozygosity track
    • CNV results report
    • CNV algorithm report
  • Structural Variant Caller
    • Output from the Structural Variant Caller
  • Target Region Coverage Analysis
    • Output from Target Region Coverage Analysis
• Batching workflows
• Legacy tools
  • Detect Fusion Genes
  • Refine Fusion Genes
• Appendices
  • Install and uninstall plugins
    • Installation of plugins
    • Uninstalling plugins
• Bibliography

Create Consensus Sequences from Variants

Using the Create Consensus Sequences from Variants tool, consensus sequences can be created from a Variant track () and the matching reference genome (). In addition, it is an option to mask out low coverage regions () with N's when a coverage track is provided.

Note: To create a low coverage region track use the Create Mapping Graph () tool to identify coverage in your sample followed by filtering on specified frequency using the Identify Graph Threshold Areas () tool.

The Create Consensus Sequences from Variants tool offers a number of filtering options:

• Minimum frequency for inclusion - Include variants above a selected frequency in the consensus.
• Ambiguity threshold - Mask out positions where the most commonly observed nucleotide is seen in fewer than the specified fraction of reads.
• Ignore frameshift variants - Filter out Indels of size 1,2,4,5 etc. A minimum frequency can be specified for when to include a variant.

Running the Create Consensus Sequences from Variants tool

To run the Create Consensus Sequences from Variants tool, go to:

        Toolbox | Resequencing Analysis () | Create Consensus Sequences from Variants ()

In the first step, select the variant track that contains the variants from which the consensus should be created.

The next step provides options for how to construct the consensus sequences (see figure 24.1).

Figure 24.1: Options available to configure when running the Create Consensus Sequences from Variants tool.

In the Tracks parameters, specify the reference genome and optionally provide an annotation track containing low coverage regions.

Variant handling parameters include:

• Minimum frequency for inclusion ranging from 0.0 to 1.0 (Default 0.8).
• Ambiguity threshold for masking variants with N's range from 0.0 to minimum frequency for inclusion (Default is 0.5). When generating the consensus, positions with variants that have a frequency between the ambiguity threshold and the minimum frequency for inclusion are replaced with N's. Positions with variants below the ambiguity threshold use the nucleotide from the reference. Note: only none-Indel variants can be masked using this threshold.
• Ignore frameshift variants. Enabled when ticked, otherwise a frequency can be specified in order to include frameshift at a certain frequency (default is 1.0). Note, frameshift variants are here defined to cover Indels not fitting with a 3 codon base structure in its simplest form. This feature is especially relevant for Virus consensus creation where frameshift variants are unlikely.

In the final step, specify the output type and a save data location.

The tool offers two types of consensus sequence format:

• Sequence Track A Consensus Genome Track.
• Sequence List A Consensus Sequence list.

To get an overview of the variants and masking in the consensus sequences an option for smaller genomes is to map the Consensus Sequence List against the reference sequence using Map Reads to Reference () and look at the Sample mapping and table. An example for the SARS-CoV-2 (MN908947.3) consensus and reference is given in figure 24.2 and 24.3).

Figure 24.2: Alignment of a mapped SARS-CoV-2 consensus against MN908947.3 reference genome.

Figure 24.3: Variant representation of the alignment shown in figure 24.2

The Map Long Read to Reference () tool should be used for bigger genomes. The Map Long Read to Reference () is part of the Long Read Support Plugin and can be downloaded from the Plugins Manager (see http://resources.qiagenbioinformatics.com/manuals/longreadsupport/current/index.php?manual=Map_Long_Reads_Reference.html for further details).

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