ALT-Aware Mapping

The GRCh38 human reference contains many more alternate haplotypes (ALT contigs) than previous versions of the reference. Generally, including ALT contigs in the mapping reference improves mapping and variant calling specificity, because misalignments are eliminated for reads matching an ALT contig but scoring poorly against the primary assembly. However, mapping with GRCh38’s ALT contigs without special treatment can substantially degrade variant calling sensitivity in corresponding regions, because many reads align equally well to an ALT contig and to the corresponding position in the primary assembly. DRAGEN ALT-aware mapping eliminates this issue, and instead obtains both sensitivity and specificity improvements from ALT contigs.

ALT-aware mapping requires hash tables that are built with ALT liftover alignments specified (see ALT-Aware Hash Tables). If a hash table built with liftover alignments is provided, DRAGEN automatically runs with ALT-aware mapping. Set the --alt-aware option to false to disable ALT-Aware mapping with a liftover reference.

DRAGEN requires ALT-Aware hash tables for any hg19 or GRCh38 reference where ALT contigs are detected. To disable this requirement in DRAGEN, set the --ht-alt-aware-validate option to false.

When ALT-aware mapping is enabled, the mapper and aligner are aware of the liftover relationship between ALT contig positions and corresponding primary assembly positions. Seed matches within ALT contigs are used to obtain corresponding primary assembly alignments, even if the latter score poorly. Liftover groups are formed, each containing a primary assembly alignment candidate, and zero or more ALT alignment candidates that lift to the same location. Each liftover group is scored according to its best-matching alignments, taking properly paired alignments into account. The winning liftover group provides its primary assembly representative as the primary output alignment, with MAPQ calculated based on the score difference to the second-best liftover group. Emitting primary alignments within the primary assembly maintains normal aligned coverage and facilitates variant calling there. If the --Aligner.en-alt-hap-aln option is set to 1 and --Aligner.supp-aligns is greater than 0, then corresponding alternate haplotype alignments can also be output, flagged as supplementary alignments.

The following is a comparison of alternative options for dealing with alternate haplotypes.

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Mapping without ALT contigs in the reference:

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False-positive variant calls result when reads matching an alternate haplotype misalign somewhere else.

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Poor mapping and variant calling sensitivity where reads matching an ALT contig differ greatly from the primary assembly.

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Mapping with ALT contigs but no ALT awareness:

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False-positive variant calls from misaligned reads matching ALT contigs are eliminated.

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Low or zero aligned coverage in primary assembly regions covered by alternate haplotypes, due to some reads mapping to ALT contigs.

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Low or zero MAPQ in regions covered by alternate haplotypes, where they are similar or identical to the primary assembly.

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Variant calling sensitivity is dramatically reduced throughout regions covered by alternate haplotypes.

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Mapping with ALT contigs and alt awareness:

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False-positive variant calls from misaligned reads matching ALT contigs are eliminated.

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Normal aligned coverage in regions covered by alternate haplotypes because primary alignments are to the primary assembly.

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Normal MAPQs are assigned because alignment candidates within a liftover group are not considered in competition.

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Good mapping and variant calling sensitivity where reads matching an ALT contig differ greatly from the primary assembly.