DNA Mapping

DNA Mapping

Seed Density Option

The seed-density option controls how many (normally overlapping) primary seeds from each read the mapper looks up in its hash table for exact matches. The maximum density value of 1.0 generates a seed starting at every position in the read, ie, (L-K+1) K-base seeds from an L-base read.

Seed density must be between 0.0 and 1.0. Internally, an available seed pattern equal or close to the requested density is selected. The sparsest pattern is one seed per 32 positions, or density 0.03125.

• Accuracy Considerations--Generally, denser seed lookup patterns improve mapping accuracy. However, for modestly long reads (eg, 50 bp+) and low sequencer error rates, there is little to be gained beyond the default 50% seed lookup density.

• Speed Considerations--Denser seed lookup patterns generally slow down mapping, and sparser seed patterns speed it up. However, when the seed mapping stage can run faster than the aligning stage, a sparser seed pattern does not make the mapper much faster.

Relationship to Reference Seed Interval

Functionally, a denser or sparser seed lookup pattern has an impact very similar to a shorter or longer reference seed interval (build hash table option --ht-ref-seed-interval). Populating 100% of reference seed positions and looking up 50% of read seed positions has the same effect as populating 50% of reference seed positions and looking up 100% of read seed positions. Either way, the expected density of seed hits is 50%.

More generally, the expected density of seed hits is the product of the reference seed density (the inverse of the reference seed interval) and the seed lookup density. For example, if 50% of reference seeds are populated and 33.3% (1/3) of read seed positions are looked up, then the expected seed hit density should be 16.7% (1/6).

DRAGEN automatically adjusts its precise seed lookup pattern to ensure it does not systematically miss the seed positions populated from the reference. For example, the mapper does not look up seeds matching only odd positions in the reference when only even positions are populated in the hash table, even if the reference seed interval is 2 and seed-density is 0.5.

Map Orientations Option

The --Mapper.map-orientations option is used in mapping reads for bisulfite methylation analysis. It is set automatically based on the value set for ‑‑methylation-protocol.

The --Mapper.map-orientations option can restrict the orientation of read mapping to only forward in the reference genome, or only reverse-complemented. The valid values for --map-orientations are as follows.

• 0--Either orientation (default)

• 1--Only forward mapping

• 2--Only reverse-complemented mapping

If mapping orientations are restricted and paired end reads are used, the expected pair orientation can only be FR, not FF or RF.

Seed-Editing Options

Although DRAGEN primarily maps reads by finding exact reference matches to short seeds, it can also map seeds differing from the reference by one nucleotide by also looking up single-SNP edited seeds. Seed editing is usually not necessary with longer reads (100 bp+), because longer reads have a high probability of containing at least one exact seed match. This is especially true when paired ends are used, because a seed match from either mate can successfully align the pair. But seed editing can, for example, be useful to increase mapping accuracy for short single-ended reads, with some cost in increased mapping time. The following options control seed editing:

Seed Editing Options

Command-Line Option Name	Configuration File Option Name
--Mappper.seed-density	seed-density
-Mapper.edit-mode	edit-mode
--Mapper.edit-seed-num	edit-seed-num
--Mapper.edit-read-len	edit-read-len
--Mapper.edit-chain-limit	edit-chain-limit

edit-mode and edit-chain-limit

The edit-mode and edit-chain-limit options control when seed editing is used. The following four edit-mode values are available:

Mode	Description
0	No editing (default)
1	Chain length test
2	Paired chain length test
3	Full seed editing

Edit mode 0 requires all seeds to match exactly. Mode 3 is the most expensive because every seed that fails to match the reference exactly is edited. Modes 1 and 2 employ heuristics to look up edited seeds only for reads most likely to be salvaged to accurate mapping.

The main heuristic in edit modes 1 and 2 is a seed chain length test. Exact seeds are mapped to the reference in a first pass over a given read, and the matching seeds are grouped into chains of similarly aligning seeds. If the longest seed chain (in the read) exceeds a threshold edit-chain-limit, the read is judged not to require seed editing, because there is already a promising mapping position.

Edit mode 1 triggers seed editing for a given read using the seed chain length test. If no seed chain exceeds edit-chain-limit (including if no exact seeds match), then a second seed mapping pass is attempted using edited seeds. Edit mode 2 further optimizes the heuristic for paired-end reads. If either mate has an exact seed chain longer than edit-chain-limit, then seed editing is disabled for the pair, because a rescue scan is likely to recover the mate alignment based on seed matches from one read. Edit mode 2 is the same as mode 1 for single-ended reads.

edit-seed-num and edit-read-len

For edit modes 1 and 2, when the heuristic triggers seed editing, these options control how many seed positions are edited in the second pass over the read. Although exact seed mapping can use a densely overlapping seed pattern, such as seeds starting at 50% or 100% of read positions, most of the value of seed editing can be obtained by editing a much sparser pattern of seeds, even a nonoverlapping pattern. Generally, if a user application can afford to spend some additional amount of mapping time on seed editing, a greater increase in mapping accuracy can be obtained for the same time cost by editing seeds in sparse patterns for a large number of reads, than by editing seeds in dense patterns for a small number of reads.

Whenever seed editing is triggered, these two options request edit-seed-num seed editing positions, distributed evenly over the first edit-read-len bases of the read. For example, with 21-base seeds, edit-seed-num=6 and edit-read-len=100, edited seeds can begin at offsets {0, 16, 32, 48, 64, 80} from the 5' end, consecutive seeds overlapping by 5 bases. Because sequencing technologies often yield better base qualities nearer the (5') beginning of each read, this can focus seed editing where it is most likely to succeed. When a particular read is shorter than edit-read-len, fewer seeds are edited.

Seed editing is more expensive when the reference seed interval (build hash table option ‑-ht‑ref-seed-interval) is greater than 1. For edit modes 1 and 2, additional seed editing positions are automatically generated to avoid missing the populated reference seed positions. For edit mode 3, the time cost can increase dramatically because query seeds matching unpopulated reference positions typically miss and trigger editing.

DNA Aligning

Smith-Waterman Alignment Scoring Settings

The first stage of mapping is to generate seeds from the read and look for exact matches in the reference genome. These results are then refined by running full Smith-Waterman alignments on the locations with the highest density of seed matches. This well-documented algorithm works by comparing each position of the read against all the candidate positions of the reference. These comparisons correspond to a matrix of potential alignments between read and reference. For each of these candidate alignment positions, Smith-Waterman generates scores that are used to evaluate whether the best alignment passing through that matrix cell reaches it by a nucleotide match or mismatch (diagonal movement), a deletion (horizontal movement), or an insertion (vertical movement). A match between read and reference provides a bonus, on the score, and a mismatch or indel imposes a penalty. The overall highest scoring path through the matrix is the alignment chosen.

The specific values chosen for scores in this algorithm indicate how to balance, for an alignment with multiple possible interpretations, the possibility of an indel as opposed to one or more SNPs, or the preference for an alignment without clipping. The default DRAGEN scoring values are reasonable for aligning moderate length reads to a whole human reference genome for variant calling applications. But any set of Smith-Waterman scoring parameters represents an imprecise model of genomic mutation and sequencing errors, and differently tuned alignment scoring values can be more appropriate for some applications.

The following alignment options control Smith-Waterman Alignment:

Command-Line Option Name	Configuration File Option Name
--Aligner.global	global
--Aligner.match-score	match-score
--Aligner.match-n-score	match-n-score
--Aligner.mismatch-pen	mismatch-pen
--Aligner.gap-open-pen	gap-open-pen
--Aligner.gap-ext-pen	gap-ext-pen
--Aligner.unclip-score	unclip-score
--Aligner.no-unclip-score	no-unclip-score
--Aligner.aln-min-score	aln-min-score
--Aligner.min-score-coeff	min-score-coeff

• global The global option (value can be 0 or 1) controls whether alignment is forced to be end-to-end in the read. When set to 1, alignments are always end-to-end, as in the Needleman-Wunsch global alignment algorithm (although not end-to-end in the reference), and alignment scores can be positive or negative. When set to 0, alignments can be clipped at either or both ends of the read, as in the Smith-Waterman local alignment algorithm, and alignment scores are nonnegative. Generally, global=0 is preferred for longer reads, so significant read segments after a break of some kind (large indel, structural variant, chimeric read, and so forth) can be clipped without severely decreasing the alignment score. Setting global=1 might not have the desired effect with longer reads because insertions at or near the ends of a read can function as pseudoclipping. Also, with global=0, multiple (chimeric) alignments can be reported when various portions of a read match widely separated reference positions. Using global=1 is sometimes preferable with short reads, which are unlikely to overlap structural breaks, unable to support chimeric alignments, and are suspected of incorrect mapping if they cannot align well end-to-end. Consider using the unclip-score option, or increasing it, instead ofsetting global=1, to make a soft preference for unclipped alignments.

• match-score The match-score option specifies the score for a read nucleotide matching a reference nucleotide (A, C, G, or T), or matching a reference 2–3 nucleotide IUPAC-IUB code. Its value is an unsigned integer, from 0 to 15. match_score=0 can only be used when global=1. A higher match score results in longer alignments, and fewer long insertions.

• match-n-score The match-n-score option specifies the score for an aligned position where the read position and/or the reference position is an N code. This option is a signed integer, from -16 to 15.

• mismatch-pen The mismatch-pen option is the penalty (negative score) for a read nucleotide mismatching any reference nucleotide or IUPAC-IUB code, except N. This option is an unsigned integer, from 0 to 63. A higher mismatch penalty results in alignments with more insertions, deletions, and clipping to avoid SNPs.

• gap-open-pen The gap-open-pen option is the penalty (negative score) for opening a gap (ie, an insertion or deletion). This value is only for a 0-base gap. It is always added to the gap length times gap-ext-pen. This option is an unsigned integer, from 0 to 127. A higher gap open penalty causes fewer insertions and deletions of any length in alignment CIGARs, with clipping or alignment through SNPs used instead.

• gap-ext-pen The gap-ext-pen option is the penalty (negative score) for extending a gap (ie, an insertion or deletion) by one base. This option is an unsigned integer, from 0 to 15. A higher gap extension penalty causes fewer long insertions and deletions in alignment CIGARs, with short indels, clipping, or alignment through SNPs used instead.

• unclip-score The unclip-score option is the score bonus for an alignment reaching the beginning or end of the read. An end-to-end alignment receives twice this bonus. This option is an unsigned integer, from 0 to 127. A higher unclipped bonus causes alignment to reach the beginning and/or end of a read more often, where this can be done without too many SNPs or indels. A nonzero unclip-score is useful when global=0 to make a soft preference for unclipped alignments. Unclipped bonuses have little effect on alignments when global=1, because end-to-end alignments are forced anyway (although 2 × unclip-score does add to every alignment score unless no-unclip-score = 1). Note that, especially with longer reads, setting unclip-score much higher than gap-open-pen can have the undesirable effect of insertions at or near one end of a read being utilized as pseudoclipping, as happens with global=1

• no-unclip-score The no-unclip-score option can be 0 or 1. The default is 1. When no-unclip-score is set to 1, any unclipped bonus (unclip-score) contributing to an alignment is removed from the alignment score before further processing, such as comparison with aln-min-score, comparison with other alignment scores, and reporting in AS or XS tags. However, the unclipped bonus still affects the best-scoring alignment found by Smith-Waterman alignment to a given reference segment, biasing toward unclipped alignments When unclip-score > 0 causes a Smith-Waterman local alignment to extend out to one or both ends of the read, the alignment score stays the same or increases if no-unclip-score=0, whereas it stays the same or decreases if no-unclip-score=1. The default, no-unclip-score=1, is recommended when global=1, because every alignment is end-to-end, and there is no need to add the same bonus to every alignment. When changing no-unclip-score, consider whether aln-min-score should be adjusted. When no-unclip-score=0, unclipped bonuses are included in alignment scores compared to the aln-min-score floor, so the subset of alignments filtered out by aln-min-score can change significantly with no-unclip-score.

• aln-min-score The aln-min-score option specifies a minimum acceptable alignment score. Any alignment results scoring lower are discarded. Increasing or decreasing aln-min-score can reduce or increase the percentage of reads mapped. This option is a signed integer (negative alignment scores are possible with global=0). aln-min-score also affects MAPQ estimates. The primary contributor to MAPQ calculation is the difference between the best and second-best alignment scores. A read's best alignment score is saved in the AS SAM tag, and the second-best score (if available) is saved in the XS tag. aln-min-score serves as the suboptimal alignment score if nothing higher was found except the best score. Therefore, increasing aln-min-score can decrease reported MAPQ for some low-scoring alignments. You can use the min-score-coeff option to adjust aln-min-score as a function of read length.

• min-score-coeff The min-score-coeff option makes adjustments to aln-min-score per read base. When using the min-score-coeff and aln-min-score options together, you can define the minimum alignment score for each read as an affine function of read lengths. The minimum score for an N-base read is calculated as follows: (min-score-coeff)\*N+(aln-min-score) The min-score-coeff option is an integer ranging from –64 to 63.999. If the value is 0, then the minimum alignment score is fixed at aln-min-score for all read length. You can use positive values for min-score-coeff to allow shorter reads to match with lower alignment scores, but require longer reads to achieve higher scores.

Paired-End Options

DRAGEN can process paired-end data passed via a pair of FASTQ files or in a single interleaved FASTQ file. The hardware maps the two ends separately, and then determines a set of alignments that seem most likely to form a pair in the expected orientation and having roughly the expected insert size. The alignments for the two ends are evaluated for the quality of their pairing, with larger penalties for insert sizes far from the expected size. The following options control processing of paired-end data:

• Reorientation The pe-orientation option specifies the expected paired-end orientation. Only pairs with this orientation can be flagged as proper pairs. Valid values are as follows:

  • 0--FR (default)

  • 1--RF

  • 2--FF

• unpaired-pen For paired end reads, best mapping positions are determined jointly for each pair, according to the largest pair score found, considering the various combinations of alignments for each mate. A pair score is the sum of the two alignment scores minus a pairing penalty, which estimates the unlikelihood of insert lengths further from the mean insert than this aligned pair. The unpaired-pen option specifies how much alignment pair scores should be penalized when the two alignments are not in properly paired position or orientation. This option also serves as the maximum pairing penalty for properly paired alignments with extreme insert lengths. The unpaired-pen option is specified in Phred scale, according to its potential impact on MAPQ. Internally, it is scaled into alignment score space based on Smith-Waterman scoring parameters.

• pe-max-penalty

The pe-max-penalty option limits how much the estimated MAPQ for one read can increase because its mate aligned nearby. A paired alignment is never assigned MAPQ higher than the MAPQ that it would have received mapping single-ended, plus this value. By default, pe-max-penalty = mapq-max = 255, effectively disabling this limit. The key difference between unpaired-pen and pe-max-penalty is that unpaired-pen affects calculated pair scores and thus which alignments are selected and pe-max-penalty affects only reported MAPQ for paired alignments.

Mean Insert Size Detection

When working with paired-end data, DRAGEN must choose among the highest-quality alignments for the two ends to try to choose likely pairs. To make this choice, DRAGEN uses a skew normal insert model to evaluate the likelihood that a pair of alignments constitutes a pair. This model is based on the observation that common library preparation methods have insert-size distributions that are sometimes close to normal, but also sometimes clearly asymmetric, often skewing toward longer insert sizes. The skew normal insert model is used only for the DNA mode.

If you know the statistics of your library prep for an input file (and the file consists of a single read group), you can specify the characteristics of the insert-length distribution: mean, standard deviation, shape (or skewness) and three quartiles. These characteristics can be specified with the Aligner.pe-stat-mean-insert, Aligner.pe-stat-stddev-insert, Aligner.pe-stat-shape-insert, Aligner.pe-stat-quartiles-insert, and Aligner.pe-stat-mean-read-len options. However, it is typically preferable to allow DRAGEN to detect these characteristics automatically.

Dragen automatically samples the insert-length distribution. When the software starts execution, it runs a sample of up to 2,000,000 pairs through the aligner, calculates the distribution, and then uses the resulting statistics for evaluating all pairs in the input set.

The DRAGEN host software reports the statistics in its stdout log in a report, as follows:

Initial paired-end statistics detected for read group RGID, based on 39042 high quality pairs for FR orientation
        Quartiles (25 50 75) = 398 409 420
        Mean = 410.192
        Standard deviation = 14.1254
        NOTE: DRAGEN's insert estimates include corrections for clipping (so they are not identical to TLEN)

        Skew-normal insert distribution applied:
          Position (xi) = 424.084
          Scale (omega) = 19.8719
          Shape (alpha) = -1.88125

        To rerun with identical insert stats, specify:
          --Aligner.pe-stat-mean-insert=424.084
          --Aligner.pe-stat-stddev-insert=19.8719
          --Aligner.pe-stat-shape-insert=-1.88125
          --Aligner.pe-stat-quartiles-insert="398 409 420"
          --Aligner.pe-stat-mean-read-len=101

Note that the Mean, Standard deviation and Quartiles reported above are the sample mean, standard deviation and quartiles calculated from the initial sample of up to 2,000,000 pairs, assuming a normal distribution. The sample mean and standard deviation are used to fit the parameters of a skew-normal distribution. A skew-normal distribution is defined by starting with an underlying normal distribution (whose mean we call position or xi and standard deviation we call scale or omega) and folding a varying portion of the probability mass from one side of the mean (e.g., left side) to the other (e.g., right) side. The portion folded varies smoothly, from 0% at the original mean, approaching 100% from the left tail to the right tail. A shape parameter which we call alpha controls how rapidly the folded fraction increases, and at alpha=0 there is no folding and the distribution remains normal.

In the standard output, we also include the command line options needed to reproduce the DRAGEN run with the same insert stat settings. Note that when specifying stats on the command line, the skew-normal xi value should be used for Aligner.pe-stat-mean-insert. The omega value should be used for Aligner.pe-stat-stddev-insert, and the alpha value should be used for Aligner.pe-stat-shape-insert. If Aligner-pe-stat-shape-insert is not specified on the command line, a default value of 0 is assumed.

The insert length distribution for each sample is written to fragment_length_hist.csv. Each sample starts with the following lines

 #Sample: sample name
 FragmentLength,Count

These lines are followed by the histogram for the first ~2M read pairs for DNA (~100K read pairs for RNA). The histogram counts are aggregated across all read groups sharing the same sample id (RGSM field).

When the number of sample pairs is very small, there is not enough information to characterize the distribution with high confidence. In this case, DRAGEN applies default statistics that specify a very wide insert distribution, which tends to admit pairs of alignments as proper pairs, even if they may lie tens of thousands of bases apart. In this situation, DRAGEN outputs a message, as follows:

WARNING: Less than 28 high quality pairs found - standard deviation is
calculated from the small samples formula

The small samples formula calculates standard deviation as follows:

 if samples < 3 then                                                     
      standard deviation = 10000                                          
 else if samples < 28 then                                               
    standard deviation = 25 * (standard deviation + 1) / (samples - 2) 
 end if                                                                   
                                                                          
 if standard deviation < 12 then                                         
      standard deviation = 12                                             
 end if                                                                   

The default model is "standard deviation = 10000". If the first 2M reads are unmapped or if all pairs are improper pairs, then the standard deviation is set to 10000 and the mean and quartiles are set to 0. Note that the minimum value for standard deviation is 12, which is independent of the number of samples. Also, in the DNA mode when we have fewer than 1000 high quality alignments we revert to the normal distribution based insert model, because of insufficient number of samples to accurately estimate the parameters of the skew normal distribution.

For RNA-Seq data, the insert size distribution is not normal due to pairs containing introns. The DRAGEN software estimates the distribution using a kernel density estimator to fit a long tail to the samples. This estimate leads to a more accurate mean and standard deviation for RNA-Seq data and proper pairing.

DRAGEN writes detected paired-end stats into a tab-delimited log file in the output directory called .insert-stats.tab. This file contains the statistical distribution of detected insert sizes for each read group, including quartiles, mean, standard deviation, shape, minimum, and maximum. The information matches the standard-out report above. Additionally, the log file includes the minimum and maximum insert limits that DRAGEN applied for rescue scans. Note that the reported mean and standard deviation in this tab-limited log file are the xi and omega parameters of the skew-normal distribution.

Rescue Scans

For paired-end reads, where a seed hit is found for one mate but not the other, rescue scans hunt for missing mate alignments within a rescue radius of the mean insert length. Normally, the DRAGEN host software sets the rescue radius to 2.5 standard deviations of the empirical insert distribution. But in cases where the insert standard deviation is large compared to the read length, the rescue radius is restricted to limit mapping slowdowns. In this case, a warning message is displayed, as follows:

Rescue radius = 220
     Effective rescue sigmas = 0.5
            WARNING: Default rescue sigmas value of 2.5 was overridden by host software!
            The user may wish to set rescue sigmas value explicitly with --Aligner.rescue-sigmas

Although the user can ignore this warning, or specify an intermediate rescue radius to maintain mapping speed, it is recommended to use 2.5 sigmas for the rescue radius to maintain mapping sensitivity. To disable rescue scanning, set max-rescues to 0.

Output Options

DRAGEN can track multiple independent alignments for each read. These alignments include the optimal (primary) one, as well as those mapping different subsegments of the read, (chimeric/supplementary), and sub-optimal (secondary) mappings of the read to different areas of the reference.

For DNA alignment by default, DRAGEN can emit one primary alignment for each read, up to three chimeric alignments (Aligner.supp-aligns=3), and no secondary alignments (Aligner.sec-aligns=0). The maximum user-specified value for supp-aligns or sec-aligns is 4095.

You can use the following configuration options to control how many of each type of alignment to include in DRAGEN output.

• mapq-max The mapq-max option specifies a ceiling on the estimated MAPQ that can be reported for any alignment, from 0 to 255. If the calculated MAPQ is higher, this value is reported instead. The default is 60.

• supp-aligns, sec-aligns The supp-aligns and sec-aligns options restrict the maximum number of supplementary (ie, chimeric and SAM FLAG 0x800) alignments and secondary (ie, suboptimal and SAM FLAG 0x100) alignments, respectively, that can be reported for each read. A maximum of 4095 supplementary alignments and 4095 secondary alignments can be reported for any read, in addition to a primary alignment. High settings for these two options impact speed so it is advisable to increase only as needed.

• sec-phred-delta The sec-phred-delta option controls which secondary alignments are emitted based on the alignment score relative to the primary reported alignment. Only secondary alignments with likelihood within this Phred value of the primary are reported.

• sec-aligns-hard The sec-aligns-hard option suppresses the output of all secondary alignments if there are more secondary alignments than can be emitted. Set sec-aligns-hard to 1 to force the read to be unmapped when not all secondary alignments can be output.

• supp-as-sec When the supp-as-sec option is set to 1, then supplementary (chimeric) alignments are reported with SAM FLAG 0x100 instead of 0x800. The default is 0. The supp-as-sec option provides compatibility with tools that do not support FLAG 0x800.

• hard-clips The hard-clips option is used as a field of 3 bits, with values ranging from 0 to 7. The bits specify alignments, as follows:

  • Bit 0--primary alignments

  • Bit 1--supplementary alignments

  • Bit 2--secondary alignments

Each bit determines whether local alignments of that type are reported with hard clipping (1) or soft clipping (0). The default is 6, meaning primary alignments use soft clipping and supplementary and secondary alignments use hard clipping.

Mapping with ALT-contigs

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.

Masked Based ALT-awareness

The recomeneded and default approach for dealing with ALT-contigs in DRAGEN is masking regions of ALT contigs of high similarity to their corresponding primary contig. This approach is more accurate than liftover based ALT-awarness because there are many places where the "correct" or most useful liftover between a long ALT haplotype and the primary assembly is ambiguous. Incorrect liftover can produce dense clusters of mismapped reads and false variant calls. The base masking approach has the benefits of using ALT contigs without the negative consequences.

Masked hash tables are built from a standard hg18 or hg38 FASTA that contains ALT contigs. The hash table builder will automatically mask regions of the ALT contigs with Ns.

Liftover Based ALT-awarness

With liftover based ALT-awareness, 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.

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.

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

• Mapping without ALT contigs in the reference:

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

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

• Mapping with ALT contigs but no ALT awareness:

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

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

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

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

• Mapping with ALT contigs and ALT awareness:

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

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

  • Normal MAPQs are assigned because alignment candidates in alternative haplotypes are not considered in competition.

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

DRAGEN Multigenome Mapper

The Multigenome Mapper in DRAGEN significantly improves the accuracy of mapping Illumina reads, particularly in challenging regions such as segmental duplications and other difficult to map regions. This advanced method leverages population haplotypes from pangenome references to incorporate additional variant information, constructing alternative haplotype paths that improve reads mapping. By offering these alternate paths, the Multigenome Mapper enables reads containing population-specific variants to align directly to their most likely genomic locations, reducing mapping ambiguity. This improved mapping also results in improved variant calling accuracy.

When given a set of population variants (VCF) or haplotypes, the pangenome reference modification is categorized in the following types:

• Alternate contigs represent population haplotypes. Alt-contigs can have a single variant or a combination of nearby phased variants.

• Ambiguous codes (IUPAC codes) to represent SNPs. To improve alignment, it edits the reference FASTA with isolated population SNPs.

• Haplotype database. An additional haplotype database is built and used to augment the reference FASTA with population variants. A multigenome based mapper algorithm is used to score read alignment according to the variants in this database.

The DRAGEN pangenome hashtables are available to download from the DRAGEN Software Support Site page.