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Alignment

During the alignment step, the banded Smith-Waterman algorithm aligns clusters from each sample against amplicon sequences specified in the manifest file.

The banded Smith-Waterman algorithm performs sequence alignments to determine similar regions between two sequences. Instead of comparing the total sequence, the Smith-Waterman algorithm compares segments of all possible lengths. Local alignments are useful for dissimilar sequences that are suspected to contain regions of similarity within the larger sequence. This process allows alignment across small amplicon targets, often less than 10 bp.

This assay sequences DNA using paired-end reads. Coverage is reported in two forms: per-amplicon and per-base. For per-amplicon coverage, ‘read’ coverage is reported. This form causes each read in a pair to be counted separately, and the total number of reads that are assigned to the amplicon are reported. For per-base coverage, ‘collapsed read’ or ‘fragment’ coverage is reported, where the reads in a pair are combined where they overlap. This type of read causes coverage to be counted only one time per pair. The information provides a more robust view of the data, as it allows combining two observations of the same underlying data (ie, a molecular fragment).

Each paired-end read is evaluated in terms of its alignment to the relevant probe sequences for that read.

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Read 1 is evaluated against the reverse complement of the Downstream Locus-Specific Oligos (DLSO).

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Read 2 is evaluated against the Upstream Locus-Specific Oligos (ULSO).

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If the start of a read matches a probe sequence with no more than one mismatch, the full length of the read is aligned against the amplicon target for that sequence.

Alignments that include more than three indels are filtered from alignment results. Filtered alignments are written in alignment files as unaligned and are not used in variant calling.

NOTE

Indel size is defined within the context of an individual amplicon. For example, a 16 bp insertion, followed by a 10 bp insertion in the same read is considered 26 bp in the maximum index size parameter. By default, the TruSight Tumor 15 analysis module is configured to report indels with a maximum size of 25 bp. You can optionally configure the maximum index size to report larger detected indels. The maximum index size can be set from 25 bp to 70 bp.

Due to the way genes are tiled by amplicons, indels larger than 25 bp may not be detected as they may overlap amplicon boundaries.

For Research Use Only. Not for use in diagnostic procedures.