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• Introduction
  • Contact information
  • System requirements
  • Installing modules
    • Licensing modules
    • Uninstalling modules
  • Installing server extensions
    • Licensing server extensions
• Methods and tools
  • Methods
    • Trimming
    • Readmapping
    • Deduplication
    • Local realignment
    • Structural variant detection
    • UMI grouping
    • Primer trimming
    • Germline variant detection
    • Somatic variant detection
    • Tumor normal variant detection
    • Batching
    • Limitations
  • Tools
    • LightSpeed Fastq to Germline Variants
      • LightSpeed Fastq to Germline Variants outputs
    • LightSpeed Fastq to Somatic Variants
      • LightSpeed Fastq to Somatic Variants outputs
    • LightSpeed Fastq to Somatic Variants Tumor Normal
      • LightSpeed Fastq to Somatic Variants Tumor Normal outputs
    • Report from LightSpeed Fastq to Variants tools
    • Calculate TMB Score (LightSpeed)
    • Copy Number Variant Detection (WGS) (beta)
      • Copy Number Variant Detection (WGS) (beta) outputs
• Template Workflows
  • General Template Workflows
    • Fastq to Annotated Germline Variants
      • Outputs from Fastq to Annotated Germline Variants
    • Fastq to Annotated Germline Variants with Coverage Analysis
      • Outputs from Fastq to Annotated Germline Variants with Coverage Analysis
    • Fastq to Annotated Somatic Variants
      • Outputs from Fastq to Annotated Somatic Variants
    • Fastq to Annotated Somatic Variants with Coverage Analysis
      • Outputs from Fastq to Annotated Somatic Variants with Coverage Analysis
    • Fastq to Annotated Somatic Variants (Tumor Normal)
      • Outputs from Fastq to Annotated Somatic Variants (Tumor Normal)
    • Fastq to Annotated Somatic Variants (Tumor Normal) with Coverage Analysis
      • Outputs from Fastq to Annotated Somatic Variants (Tumor Normal) with Coverage Analysis
    • Fastq to Germline CNV Control
      • Outputs from Fastq to Germline CNV Control
    • Fastq to Somatic CNV Control
      • Outputs from Fastq to Somatic CNV Control
  • Template Workflows - QIAseq Targeted DNA
    • QIAseq Fastq to Annotated Germline Variants
      • Outputs from QIAseq Fastq to Annotated Germline Variants
    • QIAseq Fastq to Annotated Somatic Variants
      • Outputs from QIAseq Fastq to Annotated Somatic Variants
    • QIAseq Fastq to Germline CNV Control
      • Outputs from QIAseq Fastq to Germline CNV Control
    • QIAseq Fastq to Somatic CNV Control
      • Outputs from QIAseq Fastq to Somatic CNV Control
  • Template Workflows - QIAseq Targeted DNA Pro
    • QIAseq Pro Fastq to Annotated Germline Variants
      • Outputs from QIAseq Pro Fastq to Annotated Germline Variants
    • QIAseq Pro Fastq to Annotated Somatic Variants
      • Outputs from QIAseq Pro Fastq to Annotated Somatic Variants
    • QIAseq Pro Fastq to Germline CNV Control
      • Outputs from QIAseq Pro Fastq to Germline CNV Control
    • QIAseq Pro Fastq to Somatic CNV Control
      • Outputs from QIAseq Pro Fastq to Somatic CNV Control
• Bibliography

Limitations

Data

LightSpeed is developed for and has been optimized on Illumina paired-end short read sequencing data. Paired-end sequencing data from other platforms utilizing the same data structure and similar read lengths can be expected to perform equally well with LightSpeed unless the background error-rate is markedly different. Analysis of other types of sequencing reads may not result in similar processing times or variant calls of an equivalent quality. Reads that are longer than 800 base pairs cannot be processed.

Variant detection

The germline variant detection algorithm in LightSpeed is based on a model expecting diploid genomes. Therefore, LightSpeed cannot be expected to accurately detect germline variants in genomes with other ploidies. In addition, alternate ploidies of sex chromosomes are not considered in the variant detection algorithm.

Somatic variant detection with LightSpeed is possible for variants down to a variant allele frequency of 0.1%. Variants below this frequency will not be considered. However, in order to ensure high accuracy in variant calling, we recommend only calling variants down to a variant allele frequency of approx. 1%.

Reference sequence

LightSpeed considers all chromosomes to be linear. Hence, for read mapping, circular chromosomes are linearized with position 1 starting at the junction of the chromosome. No reads will be mapped accross the junction of circular chromosomes.

UMI grouping

• The maximum number of reads used for creating a UMI consensus read is 100,000. Therefore, UMI groups with more than 100,000 reads will be merged into more than one consensus UMI read.
• LightSpeed UMI grouping requires that reads have similar mapping positions. In data from single primer extension protocols, such as many primer based QIAseq protocols, read pairs representing the same DNA fragment with the same UMI sequence can originate from different primers. This can happen if primers in the same direction are located near each other, making it possible for a downstream primer to amplify a PCR product generated from an upstream primer. LightSpeed will not group reads originating from different primers.
• When UMIs are used to group reads, the sequence is compared base by base. If an insertion or deletion is present in the beginning of a UMI sequence, this will likely prevent the reads from being grouped because all bases after the variant will be mismatches.

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