Sorting and Duplicate Marking

Sorting

The map/align system produces a BAM file sorted by reference sequence and position by default. Creating this BAM file typically eliminates the requirement to run samtools sort or any equivalent postprocessing command. The ‑‑enable-sort option can be used to enable or disable creation of the BAM file, as follows:

• To enable, set to true.

• To disable, set to false.

On the reference hardware system, running with sort enabled increases run time for a 30x full genome by about 6--7 minutes.

Duplicate Marking

Marking or removing duplicate aligned reads is a common best practice in whole-genome sequencing. Not doing so can bias variant calling and lead to incorrect results.

The DRAGEN system can mark or remove duplicate reads, and produces a BAM file with duplicates marked in the FLAG field, or with duplicates entirely removed.

In testing, enabling duplicate marking adds minimal run time over and above the time required to produce the sorted BAM file. The additional time is approximately 1--2 minutes for a 30x whole human genome, which is a huge improvement over the long run times of open source tools.

The Duplicate Marking Algorithm

The DRAGEN duplicate-marking algorithm is modeled on the Picard toolkit's 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 read pairs are never marked as duplicates.

When DRAGEN has identified a group of duplicates, it picks one as the best of the group, and marks the others with the BAM 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 idea of this score is to try, all other things being equal, 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 winner arbitrarily.

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, and 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.

Duplicate Marking Limitations

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 (RGLB). 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.

• DRAGEN does not distinguish between optical and PCR duplicates.

Duplicate Marking Settings

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

• --enable-duplicate-marking Set to true to enable duplicate marking. When \--enable-duplicate-marking is enabled, the output is sorted, regardless of the value of the enable-sort option.

• --remove-duplicates Set to true to suppress the output of duplicate records. If set to false, set the 0x400 flag in the FLAG field of duplicate BAM records. When --remove-duplicates is enabled, then enable- duplicate-marking is forced to enabled as well.

• --dedup-min-qual Specifies the Phred quality score below which a base should be excluded from the quality score calculation used for choosing among duplicate reads.