Germline variant detection
Based on the read mapping, germline variants are identified at positions where the read alignment supports a significant difference to the reference genome.
This is achieved through a site model, where each position is first assigned a likelihood for each of the genotypes A, C, T, G, N or missing. The algorithm then iterates over the read mapping and adjusts likelihoods per position for each genotype based on observations in the data until the likelihoods no longer change. Note that broken read pairs are not considered.
Each position is then inspected, and positions where the most likely genotype(s) are different from the reference sequence are identified.
Variant types
LightSpeed Fastq to Germline Variants reports SNPs, MNVs and InDels and replacements provided that the variants are contained within at least one paired end read.
Variant annotations
Variants identified by LightSpeed Fastq to Germline Variants are annotated with the following basic information: Chromosome, Region, Type, Reference, Allele, Reference allele, Length, Zygosity, Count, Coverage, Frequency, QUAL and Genotype. Only single base pair variants, that are not adjacent to any other variants, are assigned a QUAL score.
Read about general variant annotations here: https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Variant_tracks.html.
In addition, the following LightSpeed specific annotations are available:
- Average quality The average base quality score of the bases supporting a variant. The average quality score is calculated by adding the Q scores of the nucleotides supporting the variant and dividing this sum by the number of nucleotides supporting the variant. For deletions, the average quality score reported is the lowest average quality of the two bases neighboring the deleted one. For insertions, the average quality is calculated for each of the inserted bases in the reads supporting the insertion, and the minimum of the average base qualities is reported. Average quality is only calculated for non-reference alleles, for reference alleles no average quality is reported.
- Homopolymer/STR Yes/No annotation. Yes, if the variant meets minimum repeat count, minimum repeat region length and maximum repeat element length specified in the wizard when calling variants. No, if one or more of the thresholds are not met.
- Repeat count The number of repeats excluding the variant. For example if a reference allele "AAAA" is called, and a low frequent stutter insertion allele is called "AAAAA", the repeat unit is 1 and the repeat count is 4.
- Repeat unit length The length of a repeat unit. If the repeat is a homopolymer, the unit length is 1.
- Strand balance score 1 - (p-value from binomial test given forward count, count, and forward count/coverage).
If the variants are called from UMI reads, additional UMI specific annotations will be added, see UMI grouping.