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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 local sequence alignments to determine similar regions between 2 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.
Each paired-end read is evaluated in terms of its alignment to the relevant probe sequences for that read.
| ▶ | Read 1 is evaluated against the reverse complement of the Downstream Locus-Specific Oligos (DLSO). |
| ▶ | Read 2 is evaluated against the Upstream Locus-Specific Oligos (ULSO). |
| ▶ | If the start of a read matches a probe sequence with no more than 1 mismatch, the full length of the read is aligned against the amplicon target for that sequence. |
Alignments that include more than 3 indels are filtered from alignment results. Filtered alignments are written in alignment files as unaligned and are not used in variant calling.