Limitations
Data - short reads
LightSpeed Fastq to Germline Variants, LightSpeed Fastq to Somatic Variants and LightSpeed Fastq to Somatic Variants Tumor Normal are developed for and have 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 bases cannot be processed.
Data - long reads
LightSpeed Long Reads to Germline Variants (beta) is developed for Oxford Nanopore and PacBio HiFi long reads data. 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 2,000,000 bases cannot be processed.
Variant detection
In somatic variant detection, adjacent positions exhibiting variation are grouped into clusters. Low-frequency deletions spanning multiple bases can extend such clusters. During subsequent haplotype generation, this can in rare cases lead to incorrect coverage assignment, causing SNVs that otherwise meet the candidate criteria to be excluded from consideration. As a result, these variants will not appear among the reported or filtered variants. This limitation also applies to tumor normal variant detection.
Mapping
Reads with unaligned ends that extend outside chromosomes are discarded.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 across 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.
Output naming support
The LightSpeed tools support custom names for workflow results, however, not all CLC Genomics Workbench placeholders for workflow output elements are supported. Specifically, the following are supported:- {input:1} or {2:1} The name of the first input to the workflow. This is the recommended output naming.
- {name} or {1} The default name for that output from that tool, i.e. the name that would be used if the tool was run outside a workflow context.
- {metadata} or {3} The batch unit identifier for workflows executed in batch mode. Depending on how the workflow was configured at launch, this value may be obtained from metadata. Workflows not executed in batch mode or without Iterate elements are not supported with this placeholder as the value will be identical to that substituted using {input} or {2}.
- {user} The username of the person who launched the job.
- {host} The name of the machine the job is run on.
- {year}, {month}, {day}, {hour}, {minute}, and {second} Timestamp information based on the time an output is created. Using these placeholders, items generated by a workflow at different times can have different file names.