Duplicate Marking

The DRAGEN duplicate-marking algorithm is modeled on the Picard toolkit MarkDuplicates feature. All the aligned reads are grouped into subsets in which all the members of each subset are potential duplicates.

For two pairs to be duplicates, they must have the following:

•

Identical alignment coordinates (position adjusted for soft- or hard-clips from the CIGAR) at both ends.

•

Identical orientations (direction of the two ends, with the left-most coordinate being first).

In addition, an unpaired read may be marked as a duplicate if it has identical coordinate and orientation with either end of any other read, whether paired or not.

Unmapped or read pairs are never marked as duplicates.

When DRAGEN identifies a group of duplicates, it picks one as the best of the group, and marks the others with the BAM PCR or optical duplicate flag (0x400, or decimal 1024). For this comparison, duplicates are scored based on the average sequence Phred quality. Pairs receive the sum of the scores of both ends, while unpaired reads get the score of the one mapped end. The score is intended to preserve the reads with the highest-quality base calls.

If two reads (or pairs) have exactly matching quality scores, DRAGEN breaks the tie by choosing the pair with the higher alignment score. If there are multiple pairs that also tie on this attribute, then DRAGEN chooses a pairarbitrarily.

The score for an unpaired read R is the average Phred quality score per base, calculated as follows:

Where:

•

R is a BAM record

•

QUAL is its array of Phred quality scores

•

dedup-min-qual is a DRAGEN configuration option with default value of 15.

For a pair, the score is the sum of the scores for the two ends.

This score is stored as a one-byte number, with values rounded down to the nearest one-quarter. This rounding may lead to different duplicate marks from those chosen by Picard, but because the reads were very close in quality this has negligible impact on variant calling results.

The limitations to DRAGEN duplicate marking implementation are as follows:

•

When there are two duplicate reads or pairs with very close Phred sequence quality scores, DRAGEN might choose a different winner from that chosen by Picard. These differences have negligible impact on variant calling results.

•

If using a single FASTQ file as input, DRAGEN accepts only a single library ID as a command-line argument (PGLB). For this reason, the FASTQ inputs to the system must be already separated by library ID. Library ID cannot be used as a criterion for distinguishing non-duplicates.

The following options can be used to configure duplicate marking in DRAGEN:

•

--enable-duplicate-marking

•

--remove-duplicates